IC-NRLF 


flDb 


The  ruling  ot  the  I  urck  aisc.  explanation  01  me  squares.  In 
the  first  place,  we  have  the  square  which  incloses  the  entire  ruled  sur- 
face. This  is  made  up  of  nine  squares  each  i  mm.  square.  These  are 
the  squares  to  use  in  connection  with  leukocyte  counts  with  the  white 
pipette.  They  may  be  termed  the  large  squares.  The  very  smallest 
square  which  can  be  found  are  those  made  by  the  intersection  of  the 
double-ruled  lines  in  the  center;  they  are  1/40  mm.  square  and  are 
never  used  for  any  purpose.  When  \ve  refer  to  the  small  square, 
those  which  are  found  in  groups  of  sixteen  bounded  by  double-ruled 
lines  are  intended.  These  squares  are  1/20  mm.  square  and  are  used 
in  the  count  of  red  cells.  Another  square  is  outlined  in  the  more 
sparsely  ruled  large  square  in  each  of  the  four  corners.  This  square 
includes  a  space  equal  to  sixteen  small  squares  and  in  addition  the 
spaces  in  the  frame-like  space  surrounding  make  twenty  additional 
small  squares,  cr  thirty-six  in  all.  This  space  is  not  used  ordinarily. 
DaCosta  uses  a  space  made  up  of  the  sixteen  small  squares  and  the 
bordering  upper  and  left-hand  double-line  cells.  This  gives  twenty  - 
five  small  squares. 

There  are  400  small  squares  in  each  large  square,  consequently  as 
there  are  nine  large  squares  the  entire  ruled  surface  consists  of  3600 
small  squares. 


\0  ^o   v 

V\.-va.eU><<  ^«.v. 


Principal  normal  and  pathological  blood-cells  with  average  size, 
percentage  in  a  normal  differential  count  and  the  diseases  in  which 
certain  pathological  cells  are  more  or  less  pathognomonic. 


Orv^ 
raelct 

•UO^. 

\\.t\a,eeJroc  toxxXt-t 


The  ruling  of  the  Turck  disc.  Explanation  of  the  squares.  In 
the  first  place,  we  have  the  square  which  incloses  the  entire  ruled  sur- 
face. This  is  made  up  of  nin?  squares  each  i  mm.  square.  These  are 
the  squares  to  use  in  connection  with  leukocyte  counts  with  the  white 
pipette.  They  may  be  termed  the  large  squares.  The  very  smallest 
square  which  can  be  found  are  those  made  by  the  intersection  of  the 
double-ruled  lines  in  the  center;  they  are  1/40  mm.  square  and  are 
never  used  for  any  purpose.  When  we  refer  to  the  small  square, 
those  which  are  found  in  groups  of  sixteen  bounded  by  double-ruled 
lines  are  intended.  These  squares  are  1/20  mm.  square  and  are  used 
in  the  count  of  red  cells.  Another  square  is  outlined  in  the  more 
sparsely  ruled  large  square  in  each  of  the  four  corners.  This  square 
includes  a  space  equal  to  sixteen  small  squares  and  in  addition  the 
spaces  in  the  frame-like  space  surrounding  make  twenty  additional 
small  squares,  cr  thirty-six  in  all.  This  space  is  not  used  ordinarily. 
DaCosta  uses  a  space  made  up  of  the  sixteen  small  squares  and  the 
bordering  upper  and  left-hand  double-line  cells.  This  gives  twenty- 
five  small  squares. 

There  are  400  small  squares  in  each  large  square,  consequently  as 
there  are  nine  large  squares  the  entire  ruled  surface  consists  of  3600 
small  squares. 


Wv.ova.cux' 


\0  ^o  vG  yk 


Principal  normal  and  pathological  blood-cells  with  average  size, 
percentage  in  a  normal  differential  count  and  the  diseases  in  which 
certain  pathological  cells  are  more  or  less  pathognomonic. 


The  diameter  of  the  bottom  of  this  Petri  dish  is  3  inches  or  7.5  + 
centimeters. 

The  area  of  a  circle  is  equal  to  the  square  of  the  radius  multiplied 
by  *  or  22/7. 

i  1/2  in.  =radius.  11/2x11/2=2.25.  2.25  x  22/7  =  7.07  square 
inches. 

3.75  cm.  =radius.  3.75x3.75  =  14.06.  14.06  x  22/7  =  44.1  square 
centimeters. 

Number  of  bacterial  colonies  in  i  sq.  in.  averages,  approximately, 
75.  Number  in  7.07  sq.  in.  =  530. 

Number  of  bacterial  colonies  in  i  sq.  cm.  averages,  approxi- 
mately, 12.  Number  in  44.1  sq.  cm.  =  528. 

In  a  microscopic  field,  if  the  diameter  were  8  small  squares 
(1/20  mm.),  the  radius  would  be  4  small  squares  and  the  area  of  such 
a  round  field  would  be  4  x  4  =  16  x  22/7  =  50  +  .  Such  a  field  would 
contain  50  small  squares. 


PRACTICAL  BACTERIOLOGY,  BLOOD  WORK 
AND  ANIMAL  PARASITOLOGY 

ST  I  TT 


PRACTICAL 

Bacteriology,  Blood  Work 

AND 

Animal   Parasitology 

INCLUDING 

Bacteriological    Keys,    Zoological    Tables 
and  Explanatory  Clinical  Notes 


v/V 
E.  R.  STITT,  A.  B.,  Ph.  G.,  M.  D. 

SURGEON,  u.  s.  NAVY;  GRADUATE    LONDON    SCHOOL   OF  TROPICAL   MEDICINE;   INSTRUCTOR 

IN    BACTERIOLOGY   AND    TROPICAL    MEDICINE,   U.    S.    NAVAL    MEDICAL    SCHOOL; 
LECTURER   IN    TROPICAL    MEDICINE,    JEFFERSON    MEDICAL   COLLEGE 


WITH  86  ILLUSTRATIONS 


PHILADELPHIA 

P.  BLAKISTON'S  SON  &  CO. 

1012   WALNUT   STREET 
1909 


COPYRIGHT,  1909,  BY  P.  BLAKISTON'S  SON  &  Co. 


Printed  by 

The  Maple  Press 

York,  Pa. 


PREFACE. 


WHILE  a  member  of  the  Naval  Examining  Board  and  examiner  in 
bacteriology  and  clinical  microscopy,  I  have  during  the  past  six  years 
had  an  opportunity  to  judge  of  the  qualifications  of  several  hundred 
graduates  of  the  various  medical  schools  of  the  country  from  the  stand- 
point of  practical  application  in  the  laboratory  of  that  which  they  had 
learned  as  undergraduates. 

More  particularly  I  have  made  it  a  point  to  ascertain  from  the  suc- 
cessful candidates,  while  under  instruction  at  the  Naval  Medical 
School,  the  features  of  their  laboratory  courses,  which  had  seemed  to 
them  most  practical;  such  methods  being  subsequently  tested  in  our 
own  class  work. 

As  a  result  I  have  endeavored  to  incorporate  in  this  manual  methods 
which  have  been  submitted  to  the  criticism  of  postgraduate  students 
from  all  the  leading  medical  schools  of  the  country,  and  which  have 
been  considered  by  them  adapted  to  the  requirements  of  practical, 
speedy  and  satisfactory  clinical  laboratory  diagnosis. 

For  the  laboratory  worker  the  most  valuable  asset  is  common  sense 
and  he  must  be  able  to  bring  to  mind  the  possibilities  of  the  production 
of  various  artefacts  and  results  from  trivial  errors  in  technic.  It  has 
been  my  object  to  point  out  where  such  mistakes  may  arise,  the  reasons 
for  obtaining  results  differing  from  those  ordinarily  obtained  and  the 
means  to  employ  to  eliminate  as  far  as  possible  such  results. 

We  are  too  apt  to  neglect  the  trivial  details  of  stains,  reaction  of 
media  and  the  like,  yet  it  is  only  when  every  detail  of  technic  has  been 
rigidly  carried  out  that  we  are  in  a  position  to  judge  of  the  significance 
of  an  object  observed  in  a  microscopical  preparation. 

In  bacteriology,  candidates  were  frequently  able  to  give  the  cultural 
and  morphological  characteristics  of  all  the  important  pathogenic 


VI  PREFACE. 

organisms,  yet  when  it  was  required  of  them  to  outline  the  procedure 
by  which  they  would  differentiate  members  of  the  typhoid-colon  groups 
when  encountered  in  a  plate  made  from  feces,  the  problem  appeared 
to  them  impossible.  They  possessed  the  information,  but  did  not 
know  how  to  apply  it. 

In  practical  work,  organisms  can  only  be  separated  culturally  by  the 
use  of  Keys  and  for  this  reason  Keys  are  given  at  the  beginning  of  each 
division  of  bacteria.  These  enable  one  to  quickly  place  the  organism 
isolated  in  its  respective  group.  Only  methods  of  differentiation  which 
are  applicable  in  a  physician's  private  laboratory  are  given.  Practical 
methods  for  making  the  final  identification  by  agglutination  or  other 
immunity  tests  are  described.  Technic  for  immunizing  animals  to 
furnish  such  sera  is  given  in  detail. 

The  giving  of  the  cultural  characteristics  in  a  systematic  tabulated 
Key  gives  space  in  the  notes  for  presenting  the  salient  points  in  the 
pathological  and  epidemiological  aspects  of  each  organism. 

I  have  endeavored  to  give  a  scientific  yet  practical  classification  of  the 
important  pathogenic  moulds,  a  subject  about  which  there  exists 
greater  confusion  in  the  minds  of  students  than  for  any  other.  In  the 
nomenclature,  I  have  followed  Gedoelst's  "  Les  Champignons  Para- 
sites." 

In  the  chapter  on  media  making,  it  is  believed  than  anyone  after 
reading  this  section  and  following  the  instructions  will  be  able  to  satis- 
factorily and  without  the  adjuncts  of  a  large  laboratory  make  any  kind 
of  media.  The  directions  as  to  titrations  are  given  in  detail  because 
it  is  beginning  to  be  recognized  that  reaction  of  media  in  bacteriology  is 
of  as  great  importance  as  staining  is  in  blood  work. 

The  section  on  blood  work  is  practical  and  gives  a  method  for  making 
a  Romanowsky  stain  which  is  quick  and  reliable.  The  chapter  on 
Normal  and  Pathological  Blood  gives  in  a  few  pages  the  more  impor- 
tant points  to  be  borne  in  mind  in  considering  a  possible  diagnosis. 

While  there  is  no  difference  between  the  laboratory  requirements  of 
medical  work  in  the  tropics  and  that  in  temperate  climates,  unless  by 
reason  of  such  measures  of  diagnosis  being  indispensable  in  the  tropics, 
it  has,  however,  been  my  endeavor  to  treat  every  tropical  question, 
whether  in  blood  work,  bacteriology  or  animal  parasitology,  in  a  more 


PREFACE.  Vll 

complete  way  than  is  usual  in  manuals  of  this  character.  Therefore  it 
is  believed  that  this  little  book  will  be  of  great  service  to  the  laboratory 
worker  in  the  tropics. 

It  is  only  from  working  under  Doctor  Charles  W.  Stiles  in  his  course 
of  laboratory  instruction  in  Animal  Parasitology  in  the  United  States 
Xaval  Medical  School,  that  I  feel  justified  in  presenting  a  concise  out- 
line of  the  subjects  in  medical  zoology  which  appear  to  me  to  be  most 
important  for  the  physician. 

The  system  of  arranging  tables,  showing  the  families,  genera,  etc., 
in  which  each  species  belongs  will,  it  is  believed,  greatly  simplify  the 
matter  of  classification  for  the  medical  student.  The  points  given 
under  each  parasite  are  believed  to  be  practical  ones.  When  a  parasite 
has  only  been  reported  for  man  two  or  three  times,  very  little  space  is 
given  to  it. 

Part  IV  summarizes  the  various  infections  which  may  be  found  in 
different  organs  or  excretions  of  the  body  and  embraces  both  bacterial 
and  animal  parasites.  Practical  methods  for  examining  material  are 
also  given. 

The  chapter  on  Immunity,  in  which  the  theoretical  side  is  im- 
mediately illustrated  by  the  practical  application  will  tend  to  simplify 
this  bug-bear  of  the  medical  student. 

The  illustrations  have  been  selected  with  a  view  to  bringing  out 
points  which  are  difficult  to  state  briefly  in  the  text,  and  furthermore 
they  have  been  grouped  together  so  that  comparison  of  similar  para- 
sites is  possible  without  turning  from  page  to  page. 

I  have  in  particular  to  thank  Hospital  Steward  Ebeling  of  the  Navy 
for  his  care  in  bringing  out  such  details. 

By  reason  of  the  authority  of  Braun,  it  has  been  considered  sufficient 
to  give  in  the  tables  only  the  proper  zoological  name  of  the  parasite  as 
given  in  the  1908  German  edition.  The  synonyms  have  been  omitted 
for  consideration  of  space. 

The  works  chiefly  consulted  in  addition  to  that  of  Braun  have  been : 

Allbutt's  System  of  Medicine;  Osier's  System  of  Medicine;  Muir  and 
Ritchie's  Bacteriology;  Mense's  Tropenkrankheiten;  Blanchard's  Les 
Moustiques;  Guiart  and  Grimbert's  Diagnostic;  Ehrlich's  Studies  in 
Immunity;  Stephens  and  Christopher's  Practical  Study  of  Malaria; 


vlii  PREFACE 

Daniel's  Laboratory  Studies  in  Tropical  Medicine;  Hanson's  Tropical 
Diseases;  Gedoelst's  Les  Champignons  Parasites;  Neveu-Lemaire 
Parasitologie  Humaine;  Chester's  Determinative  Bacteriology;  Leh- 
mann  and  Neumann's  Bacteriology. 

E.  R.  S. 
DECEMBER  17,  1908. 


CONTENTS. 


PART  I.     BACTERIOLOGY. 

CHAPTER  I.— APPARATUS. 

The  Microscope,  i; — Apparatus  for  sterilization,  4; — Cleaning  glassware, 
8; — Concave  slides,  fermentation  tubes.  9; — Incubators,  n; — Bacterio- 
logical pipettes,  12. 

CHAPTER  II.— CULTURE  MEDIA. 

Xutrient  bouillon,  15; — Standardization  of  reaction,  17; — Sugar-free 
bouillon,  20; — Glycerine  bouillon,  20; — Peptone  solution,  21; — Nutrient 
agar,  21; — Glycerine  agar  egg  medium,  22; — Gelatine,  22; — Litmus 
milk,  23; — Potato,  23; — Blood  serum.  24; — Blood  agar,  24; — Bile  and 
faeces  media,  25. 

CHAPTER  III.— STAINING  METHODS. 

Loffler's  methylene  blue,  28; — Carbol  fuchsin,  28; — Gram's  method,  28; — 
Acid-fast  staining,  30; — Neisser's  stain,  31; — Capsule  staining,  31; — 
Flagella  staining,  32; — Spore  staining,  32. 

CHAPTER  IV.— STUDY  AND   IDENTIFICATION  OF  BACTERIA.     GENERAL   CON- 
SIDERATIONS. 
Methods  of  isolating  bacteria,  34; — Classification,  36; — Use  of  keys,  38. 

CHAPTER  V. — STUDY  AND  IDENTIFICATION  OF  BACTERIA.     Cocci. 

Key,  41; — Streptococci,  42; — Sarcinae,  43; — Staphylococci,  44; — Pneumo- 
coccus,  46; — Gram  negative  cocci,  47. 

CHAPTER  VI.—  STUDY  AND    IDENTIFICATION    OF    BACTERLA.     SPORE-BEARING 

BACILLI. 

Key, 53;— Anthrax,  54; — Cultivation  of  anaerobes,  57; — Malignant  oedema, 
59; — B.  botulinus,  60; — B.  tetani.  62; — B.  aerogenes  capsulatus,  64. 

CHAPTER    VII. — STUDY    AND    IDENTIFICATION    OF    BACTERLA.     BRANCHING, 

CURVING  BACILLI.     MYCOBACTERIA.     CORNYEBACTERIA. 
Acid-fast    bacilli,  67; — Tubercle  bacillus,  68; — Leprosy  bacillus,  70; — 
Xon  acid-fast  branching  bacilli   71; — B.  mallei,  72; — B.  diphtheriae,  73; 
— Hofman's  bacillus,  76; — B.  xerosis,  77. 

CHAPTER  VIII.— STUDY  AND  IDENTIFICATION  OF  BACTERIA. 

Gram  negative  bacilli,  Hemophilic    bacteria,   78; — Influenza    bacillus, 
79; — Friedlander's  bacillus,  81; — Plague,  81; — Eberth,  Gartner  and  Esche- 
rich  groups,  85; — Typhoid,  86; — Dysentery  89; — Chromogenic  bacilli,  92. 
ix 


X  CONTENTS. 

CHAPTER  IX. — STUDY  AND  IDENTIFICATION  OF  BACTERIA. 
Spirilla,  94; — Cholera,  94. 

CHAPTER  X. — STUDY  AND  IDENTIFICATION  OF  MOULDS,  99. 

CHAPTER  XL — BACTERIOLOGY  OF  WATER,  AIR  AND  MILK. 
Water,  109; — Milk,  115; — Air,  117. 

CHAPTER  XII. — PRACTICAL  METHODS  IN  IMMUNITY. 

Methods  of  obtaining  immune  sera,  124;— Agglutination  tests,  126; — 
Haemolytic  experiments,  128; — Bacteriolytic  experiments,  128; — Devia- 
tion of  the  Complement,  129; — Fixation  of  the  Complement,  130; — 
Opsonic  power  and  preparation  of  vaccines,  131. 

PART  II.     STUDY  OF  THE  BLOOD. 

CHAPTER  XIII. — MlCROMETRY  AND  BLOOD  PREPARATIONS. 

Micrometry,i35; — Haemoglobin  estimation,  138; — Counting  blood,  140; — 
Study  of  fresh  blood,  145; — Blood  films,  146; — Staining  blood  films,  148; — 
lodophilia,  152. 

CHAPTER  XIV.— NORMAL  AND  PATHOLOGICAL  BLOOD. 

Color  index,  154; — Red  cells,  154; — White  cells,  156; — Eosinophilia, 
162; — Leukocytosis,  162; — Lymphocytosis,  163; — Diseases  with  a  normal 
leukocyte  count,  164; — The  primary  anaemias,  164; — Secondary  anaemias, 
166; — The  leukemias,  166. 

PART  III.     ANIMAL  PARASITOLOGY. 
CHAPTER  XV. — CLASSIFICATION  AND  METHODS,  169. 

CHAPTER  XVI.— THE  PROTOZOA. 

Rhizopoda,  173; — Flagellata,  176; — Infusoria,  183; — Sporozoa,  183; — 
The  malarial  parasite,  185. 

CHAPTER  XVII.— THE  FLAT  WORMS. 

Flukes,  194; — Liver  flukes,  195; — Intestinal  flukes,  197; — Lung  flukes, 
198; — Blood  flukes,  198; — Cestodes,  200; — Somatic  taeniasis,  205. 

CHAPTER  XVIIL— THE  ROUND  WORMS. 

Filariidae,  209; — Key  to  filarial  larvae,  213; — Trichinosis,  214; — Hook 
worms,  216; — Ascaridae,  218; — Leeches,  219. 

CHAPTER  XIX.— THE  ARACHNOIDEA. 

The  mites,  222; — The  ticks,  224; — The  Linguatulidae,  227. 

CHAPTER  XX.— THE  INSECTS. 

The  Pediculidae,  229; — The  Diptera,  231; — Fleas,  232; — Biting  flies,  234. 

CHAPTER  XXL— THE  MOSQUITOES. 

Dissection  of  the  mosquitoes,  244; — Differentiation  of  Culicinae  and 
Anophelinae,  246; — Classification  of  Culicidae,  247. 


APPENDIX.  XI 

PART  IV.     CLINICAL  BACTERIOLOGY  AND  ANIMAL 

PARASITOLOGY  OF  THE  VARIOUS   BODY 

FLUIDS  AND  ORGANS. 

CHAPTER  XXII. — DIAGNOSIS  OF  INFECTIONS  OF  THE  OCULAR  REGION,  251. 

CHAPTER  XXIII.— DIAGNOSIS  OF  INFECTIONS  OF  THE  NASAL  CAVITIES,  253. 

CHAPTER  XXIV.— EXAMINATION  OF  BUCCAL  AND  PHARYNGEAL  MATEIOAL,  255. 

CHAPTER  XXV. — EXAMINATION  OF  SPUTUM,  258. 

CHAPTER  XXVI.— THE  URINE,  261. 

CHAPTER  XXVII.— THE  RECES,  263. 

CHAPTER  XXVIII.— BLOOD  CULTURES  AND  BLOOD  PARASITES,  268. 

CHAPTER  XXIX.— THE  STOMACH  CONTENTS,  270. 

CHAPTER  XXX. — EXAMINATION  OF  Pus,  271. 

CHAPTER  XXXI.— SKIN  INFECTIONS,  273. 

CHAPTER  XXXII.— CYTODHGNOSIS,  275. 

CHAPTER  XXXIII.— RABIES,  277. 


APPENDIX. 

PREPARATION  OF  TISSUES  FOR  EXAMINATION  IN  MICROSCOPIC  SECTIONS,  279. 
MOUNTING  AND  PRESERVATION  OF  ANIMAL  PARASITES,  283. 
PREPARATION  OF  NORMAL  SOLUTIONS,  284. 
DISEASES  OF  UNKNOWN  ETIOLOGY,  285. 


BACTERIOLOGY,   BLOOD-WORK 
AND  ANIMAL  PARASITOLOGY 


CHAPTER  I. 
APPARATUS. 

THE  MICROSCOPE. 

THE  most  important  piece  of  apparatus  for  the  laboratory  worker 
is  the  microscope.  Very  satisfactory  microscopes  can  be  purchased  in 
this  country.  Instruments  of  standard  German  make  are  in  use  in 
many  laboratories  and  appear  to  give  general  satisfaction.  It  is 
impossible  to  do  good  microscopical  work  unless  the  microscope  gives 
and  continues  to  give  good  definition  and  the  working  parts  remain 
firm.  A  mechanical  stage  is  almost  a  necessity  in  connection  with 
blood-work  and  its  use  is  advantageous  in  bacterial  preparations.  For 
the  study  of  tissue  sections  the  moving  of  the  slide  with  the  fingers  is 
preferable.  Therefore,  the  mechanical  stage  should  be  capable  of 
ready  attachment  or  removal.  A  triple  or  quadruple  nose-piece, 
according  to  the  number  of  objectives  used,  is  also  indispensable. 
To  meet  the  demands  of  clinical  microscopy  there  should  be  three 
objectives,  preferably  a  16  mm.  (2/3  in.),  a  4  mm.  (1/6  in.)  and  a 
2  mm.  (1/12  in.)  homogeneous  oil  immersion.  The  Zeiss  AA  is  a 
17  mm.  objective,  and  the  Leitz  No.  3,  an  18  mm.  one.  The  Zeiss  D 
is  about  4.2  mm.  and  the  Leitz  No.  6,  a  4.4  mm.  A  dust-proof  quad- 
ruple nose-piece  with  four  objectives  will  be  found  a  great  convenience 
(in  addition  to  the  2/3-in.  and  i/i2-in.  objectives,  a  i/4-in.  for  urine  and 
blood  counting,  with  a  1/8  in.  for  examining  hanging-drop  preparations 
and  for  quick  examination  of  blood  smears).  An  apochromatic 
objective  costs  about  three  times  as  much  as  an  achromatic  one  and, 
except  in  photographic  work,  has  little  if  any  advantage. 

As  regards  oculars  (eye-pieces)  a  No.  2  and  a  No.  4  will  best  meet 
the  requirements.  For  high  magnification  a  No.  8  may  be  of  service. 


2  APPARATUS. 

The  Zeiss  oculars  are  numbered  according  to  the  amount  they  increase 
the  magnification  given  by  the  objective;  thus  a  No.  2  increases  the 
magnification,  given  by  the  objective  alone,  twice;  a  No.  8,  eight  times. 
Some  oculars  are  classified  according  to  the  equivalent  focal  distance, 
and  are  referred  to  as  i/2-in.,  i-in.  and  2-in.  oculars. 

The  oculars  in  common  use  are  known  as  negative  oculars,  by 
which  is  meant  an  ocular  in  which  the  lower  lens  (collective)  assists  in 
forming  the  real  inverted  image  which  is  focused  at  the  level  of  the 
diaphragm  writhin  the  ocular.  When  using  a  disk  micrometer,  it  is 
supported  by  this  diaphragm,  and  the  outlines  of  the  image  are  cut  by 
the  rulings  on  the  glass  disk,  and  so  we  are  enabled  to  measure  the  size 
of  the  object  being  examined.  The  measurement  of  various  bacteria, 
blood-cells  and  parasites  is  exceedingly  simple  and  assists  greatly  in  the 
study  of  bacteria,  and  is  indispensable  in  work  in  animal  parasitology. 
(For  details  of  micrometry  see  section  on  blood-work.)  When  an 
ocular  is  termed  positive,  it  refers  to  an  ocular  which  acts  as  a  simple 
microscope  in  magnifying  the  image,  the  image  being  formed  entirely 
by  the  objective  and  being  located  below  the  ocular. 

Objectives  are  usually  designated  by  their  equivalent  focal  distance. 
It  is  important  to  remember  that  the  equivalent  focal  distance  does  not 
represent  the  working  distance  of  an  objective,  by  which  is  meant  the 
distance  from  the  upper  surface  of  the  cover-glass  to  the  lower  surface 
of  the  objective.  Thus  a  i/4-in.  objective  may  have  to  be  approached 
to  the  object  so  that  the  distance  intervening  may  be  only  1/6  in.  or 
even  less.  This  explains  the  frequent  inability  to  focus  an  object  when 
a  high-power  dry  objective  (1/6  in.  or  1/8  in.)  is  used  with  a  rather 
thick  cover-glass — the  objective  possibly  having  a  short  working 
distance,  so  that  the  thickness  of  the  cover-glass  does  not  allow  of  any 
free  working  distance. 

Instrument  makers  generally  specify  the  thickness  of  cover-glass  to 
be  used  with  a  certain  tube  length,  but  as  a  practical  matter  it  will  be 
found  convenient  to  use  No.  i  (very  thin)  cover-glasses.  The  principal 
objection  to  these  is  that  they  are  more  fragile  than  a  No.  2,  but  with 
a  little  practice  in  cleaning  cover-glasses  this  is  negligible. 

One  of  the  most  fruitful  causes  of  the  crushing  of  microscopical 
.objects  and  the  overlying  cover-glass  or,  what  is  far  more  important, 


Till.    MICROSCOPE.  3 

the  breaking  <>f  the  cover-glass  of  a  hanging-drop  preparation  and 
consequent  risk  of  infection  is  the  attempt  to  focus  with  the  fine  ad- 
justment. It  should  be  an  invariable  rule  for  the  worker  to  bring  his 
objective  practically  into  contact  with  the  upper  surface  of  the  cover- 
glass,  then  using  the  coarse  adjustment  (rack  and  pinion)  to  slowly 
elevate  the  objective,  looking  through  the  eye-piece  at  the  same  time. 
In  other  words,  obtain  focus  with  the  coarse  adjustment  and  maintain 
it  with  the  fine  adjustment  (micrometer  screw).  The  fine  adjustment 
should  only  be  used  after  the  focus  is  obtained. 

It  will  be  observed  that  objectives  frequently  have  their  numerical 
aperture  marked  on  them.  This  is  expressed  by  the  letters  N.A. 
From  a  practical  stand-point  this  gives  the  relative  proportion  of  the 
rays  which  proceeding  from  an  object  can  enter  the  lens  of  the  objective 
and  form  the  image.  Of  course,  the  greater  the  number  of  rays,  the 
greater  the  N.A.,  the  better  the  definition,  and  consequently  the  better 
the  objective.  Immersion  oil,  having  the  same  index  of  refraction 
(1.52)  as  glass,  would  not  deflect  rays  coming  from  the  object  and  so 
prevent  their  entering  the  objective,  as  would  be  the  case  if  we  used  a 
dry  objective  with  an  intervening  air  .space.  In  this  case  a  portion  of 
the  rays  would  be  turned  aside  by  the  difference  in  the  refractive  index 
of  air.  As  a  rule,  the  higher  the  numerical  aperture,  the  better  the 
objective  and  the  less  the  working  distance.  In  blood  counting,  the 
cover-glass  being  comparatively  thick,  it  may  happen  that  with  a  1/6  in. 
of  high  numerical  aperture  there  may  not  be  sufficient  working  dis- 
tance to  bring  the  blood-cells  into  focus,  which  could  be  done  with  an 
objective  of  lower  numerical  aperture.  Consequently,  we  must 
always  consider  the  matter  of  working  distance  as  well  as  that  of 
numerical  aperture. 

An  important  matter  in  the  use  of  the  microscope  is  to  get  all  the 
details  possible  with  a  low  power  before  using  a  higher  power.  This, 
of  course,  does  not  apply  to  a  bacterial  preparation  where  it  is  necessary 
to  use  a  i/ 1 2-in.  or  a  high-power  dry  lens.  With  tissue  sections,  how- 
ever, it  is  not  only  advisable  to  begin  the  study  with  the  lowest  power, 
but  even  an  examination  with  the  unaided  eye  or  with  a  magnifying 
before  using  the  microscope,  will  give  a  surprising  amount  of 
information. 


4  APPARATUS. 

It  is  advisable  to  cultivate  the  use  of  both  eyes  in  doing  microscopi- 
cal work.  When  using  one  eye  the  other  should  be  kept  open  with 
accommodation  relaxed.  It  is  this  squinting  of  the  unemployed  eye 
which  so  often  fatigues.  A  strip  of  cardboard  four  or  five  inches  long, 
with  an  opening  to  fit  over  the  tube  of  the  microscope,  leaving  the  other 
end  to  block  the  vision  of  the  unused  eye,  will  prevent  the  strain. 
This  apparatus  can  be  purchased  in  vulcanite. 

Direct  sunlight  or  excessively  bright  light  is  to  be  avoided.  If 
such  conditions  must  exist  a  wrhite  shade  or  muslin  curtain  drawn 
across  the  window  is  a  necessity.  Light  from  the  north  and  from  a 
white  cloud  is  the  most  desirable.  The  technic  in  connection  with 
proper  illumination  is  probably  more  important  than  any  other  point; 
unless  the  light  is  utilized  to  the  best  advantage,  the  best  results  cannot 
be  obtained.  In  examining  fresh  blood  preparations  or  hanging  drops 
the  concave  mirror  should  be  used  and  the  light  almost  shut  off  bv  the 
iris  diaphragm  so  as  to  give  a  contour  picture.  In  examining  a 
stained  blood  or  bacterial  preparation,  the  Abbe  condenser  should  be 
properly  focused  so  as  to  best  illuminate  the  stained  film.  In  many 
instruments  set-screws  are  provided  which  check  the  elevation  of  the 
Abbe  condenser  when  the  proper  focus  is  reached.  Inasmuch  as  the 
light  from  the  condenser  should  come  to  a  focus  exactly  level  with  the 
object  studied,  it  is  evident  that  a  fixed  position  for  the  condenser  would 
not  answer  when  slides  of  different  thickness  were  used.  Always  use 
the  plane  mirror  when  examining  stained  bacterial  or  blood -films,  as 
a  color  image  is  desired.  Ordinarily  in  examining  tissue  sections,  the 
Abbe  condenser  should  either  be  put  out  of  focus  by  racking  down  or 
by  the  use  of  the  concave  mirror  and  the  narrowing  of  the  aperture  of 
the  iris  diaphragm.  Swing-out  condensers  are  now  made  which  are 
very  convenient.  The  proper  employment  of  illumination  only  comes 
with  experience,  and  one  should  continue  to  manipulate  his  mirrors, 
diaphragm  and  condenser  until  the  best  result  is  obtained.  Then 
study  the  specimen. 

APPARATUS  FOR  STERILIZATION. 

For  the  purpose  of  sterilizing  glassware,  media  and  old  cultures 
there  are  three  methods  ordinarily  employed.  The  hot-air  sterilizer, 


APPARATUS    FOR    STERILIZATMN.  5 

in  which  a  temperature  of  about  150°  C.  is  maintained  for  one  hour,  is 
ordinarily  used  for  the  sterilization  of  Petri  dishes,  test-tubes,  pipettes, 
etc.  If  the  temperature  is  allowed  to  go  too  high,  there  is  danger  of 
charring  the  cotton  plugs  and  also  of  causing  the  development  of  an 
empyreumatic  oil  which  makes  the  plugs  unsightly  and  causes  them  to 
stick  to  the  glass.  Again  we  must  be  careful  not  to  open  the  door  until 
the  temperature  has  fallen  to  60°  C.,  otherwise  here  is  danger  of 
cracking  the  glassware.  Where  gas  is  not  obtainable,  the  hot-air 
sterilizer  is  not  a  very  satisfactory  apparatus. 

The  Arnold  sterilizer  is  to  be  found  everywhere  and  can  be  used  on 
blue-flame  kerosene-oil  stoves  as  readily  as  with  gas  burners.  The 
most  convenient  form,  but  more  expensive,  is  the  Boston  Board  of 
Health  pattern.  The  ordinary  pattern,  with  a  telescoping  outer 
portion,  answers  all  purposes,  however.  In  the  Arnold,  sterilization  is 
effected  by  streaming  steam  at  100°  C.  It  is  usual  to  maintain  this 
temperature  for  fifteen  to  twenty-five  minutes  each  day  for  three  suc- 
cessive days.  The  success  of  this  procedure — fractional  sterilization 
— is  due  to  the  fact  that  many  spores  which  were  not  killed  at  the  first 
steaming  have  developed  into  vegetative  forms  within  twenty-four 
hours,  and  when  the  steam  is  then  applied  such  forms  are  destroyed. 
Experience  has  shown  that  all  the  spores  have  developed  by  the  time  of 
the  third  steaming,  so  that  with  this  final  application  of  heat  we  secure 
perfect  sterilization. 

It  is  customary  to  use  the  Arnold  for  sterilizing  gelatin  and  milk 
media,  even  when  the  autoclave  is  at  hand,  the  idea  being  that  the 
greater  heat  of  the  autoclave  may  interfere  with  the  quality  of  such 
media.  The  most  convenient  autoclave  is  the  horizontal  type,  such  as 
is  to  be  found  even-where  for  the  sterilization  of  surgical  dressings. 
The  source  of  heat  may  be  either  gas,  the  Primus  kerosene-oil  lamp  or 
steam  from  an  adjacent  boiler.  During  the  past  year,  in  the  labora- 
tory of  the  U.  S.  Xaval  Medical  School,  we  have  been  using  a  dressing 
sterilizer,  made  by  the  American  Sterilizer  Co.,  with  which  it  has  been 
possible  to  most  satisfactorily  carry  out  all  kinds  of  sterilization  thus 
doing  away  with  the  use  of  the  Arnold  and  the  hot-air  sterilizer.  It 
is  impossible  to  sterilize  ordinary  fermentation  tubes  in  the  autoclave 
on  account  of  the  boiling  up  of  the  media  and  wetting  of  the  plugs. 


APPARATUS. 


This  is  still  done  with  the  Arnold.  By  use  of  the  Durham  tubes — 
which  are  to  be  preferred,  except  for  gas  analysis — sugar  media  can  be 
thus  sterilized,  and  glassware  will  come  out  with  the  wrappers  as  dry 


C     . 


Fro.  i. — Dressing  sterilizer  showing  cylinder  containing  water  (K)  heated 
either  by  gas  or  Primus  Kerosene  lamps. 

and  the  plugs  of  test-tubes  as  stopper-like  as  could  be  effected  in  a  hot- 
air  sterilizer. 

The  objection  which  exists  in  the  use  of  some  autoclaves,  as  regards 
condensation  on  dressings  or  apparatus,  does  not  exist  in  this  type. 


STERII.I/ATloN.  7 

The  mechanism,  by  which  the  inner  and  outer  chambers  are  con- 
nected and  disconnected,  and  that  for  vacuum  production,  rest  in  the 
simple  turning  of  a  lever  from  mark  to  mark.  We  have  been  able 
with  a  gas  burner  to  obtain  a  pressure  of  fifteen  pounds  in  less  than  ten 
minutes.  In  sterilizing  test-tubes  we  place  them  in  small  rectangular 
wire  baskets,  6x5x4  ins.  These  baskets  are  to  be  preferred  to  round 
ones,  as  they  pack  more  satisfactorily  in  the  refrigerator  used  for  storing 
media.  In  sterilizing  flasks,  test-tubes,  Petri  dishes,  throat  swabs, 
pipettes,  etc.,  it  has  been  our  custom,  after  exposing  to  20  pound-  for 
twenty  minutes,  to  produce  a  vacuum  for  two  or  three  minutes;  then 
with  the  steam  in  the  outer  jacket  for  a  few  minutes  to  thoroughly  dry 
the  articles  in  the  disinfecting  chamber.  The  valve  to  the  inner 
chamber  is  then  opened  to  break  the  vacuum;  the  door  is  now  opened, 
and  the  articles  removed  in  as  dry  a  state  as  if  they  had  been  in  the 
hot-air  sterilizer. 

PRESSURE  AND  TEMPERATURE  TABLE. 

5  pounds'  pressure,  107.7°  C.,  226°  F. 

10  pounds'  pressure,  115. 5°  C.,  240°  F. 

15  pounds'  pressure,  121. 6°  C.,  250°  F. 

20  pounds'  pressure,  126. 6°  C.,  260°  F. 

25  pounds'  pressure,  130.5°  C.,  267°  F. 

30  pounds'  pressure,  134.4°  C.,  274°  F. 

All  such  articles  as  Petri  dishes,  pipettes,  swabs,  etc.  are  wrapped  in 
cheap  quality  filter-paper,  making  a  fold  and  turning  in  the  ends  as  is 
done  in  a  druggist's  package.  Old  newspapers  answer  well  for  this 
purpose.  The  sterile  swab  can  be  used  for  many  purposes  in  the 
laboratory.  They  are  most  easily  made  by  taking  a  piece  of  copper 
wire  about  eight  inches  long,  flattening  one  end  with  a  stroke  of  a 
hammer,  then  twisting  a  small  pledget  of  plain  absorbent  cotton 
around  the  flattened  end.  After  wrapping,  the  swabs  are  sterilized  in 
bunches.  We  not  only  use  them  for  getting  throat  cultures,  but  in 
addition  for  culturing  faeces,  pus  or  other  such  material.  The  pus  is 
obtained  with  a  swab,  which  material  is  then  distributed  in  a  tube  of 
sterile  bouillon  or  water.  With  the  same  swab  the  surface  of  an  agar 
plate  is  successively  stroked.  This  method  is  equally  as  satisfactory  as 


8 


APPARATUS. 


the  German  one  of  using  bent  glass  rods  for  this  purpose.  Everyone 
has  encountered  the  difficulties  attendant  upon  the  bending  of  platinum 
wires  and  also  the  possibility  of  destroying  your  organisms  by  an 
insufficiently  cooled  wire. 

CLEANING  GLASSWARE. 

It  is  a  routine  in  our  laboratory  for  everything  to  go  through  the 
sterilizer  at  125°  C.  before  anything  else  is  done.     This  is  a  safe  rule 


FIG.    2. — i,   Inoculation  of   tubes;    2,   plugging  of  tubes;    3,   filling  tubes; 
4,  Smith's  fermentation  tube;  5,  Durham's  fermentation  tube. 

when  dealing  with  dangerous  pathogenic  organisms.  As  soon  as 
taken  out  of  the  sterilizer  the  contents  are  emptied,  and  the  tubes  or 
dishes  placed  in  a  i%  solution  of  washing  soda  and  boiled.  This 
thoroughly  cleans  them.  As  the  washing  soda  slightly  raises  the 
boiling-point  and  also  makes  the  spores  more  penetrable,  it  would 
appear  that  under  ordinary  circumstances,  it  would  be  sufficient  to 
place  all  contaminated  articles  in  a  dishpan  with  the  soda  solution, 


CLEANING    GLASSWARE.  9 

and  boil  for  at  least  one  hour,  not  using  a  preliminary  sterilization  in 
the  autoclave.  The  tubes  are  now  cleaned  with  a  test-tube  brush, 
thoroughly  rinsed  with  tap  water  and  placed  in  a  i%  solution  of 
hydrochloric  acid  for  a  few  minutes;  then  rinsed  thoroughly  in 
water  and  placed  in  test-tube  baskets,  mouth  downward,  and  allowed 
to  drain  over  night.  When  thoroughly  dry  they  may  be  plugged  and 
sterilized.  To  plug  a  test-tube,  pick  out  a  little  pledget  of  plain 
absorbent  cotton  about  two  inches  in  diameter  from  a  roll.  Place 
it  over  the  center  of  the  tube  and  with  a  glass  rod  push  the  cotton  down 
the  tube  about  an  inch.  The  cleaning  fluid  commonly  used  in  labora- 
tories consists  of  one  part  each  of  potassium  bichromate  and  com- 
mercial sulphuric  acid  with  ten  parts  of  water.  This  is  an  excellent 
mixture  for  cleaning  old  slides,  etc.,  especially  when  grease  or  balsam 
is  to  be  gotten  rid  of.  It  is  very  corrosive,  however.  An  efficient  and 
less  corrosive  methcd  for  cleaning  slides  and  covei>glasses  is  to  leave 
them  over  night  in  an  acetic  acid  alcohol  mixture  (two  parts  of  glacial 
acetic  acid  to  one  hundred  parts  of  alcohol).  After  drying  and 
polishing  out  of  this  mixture,  it  is  well  to  pass  the  slides  and  cover- 
glasses  through  the  flame  of  a  Bunsen  burner  or  alcohol  lamp  to 
remove  every  vestige  of  grease.  Ordinarily,  rubbing  between  the 
thumb  and  forefinger  with  soap  and  water,  then  drying  with  an  old 
piece  of  linen,  and  finally  flaming  will  yield  a  perfect  surface  for 
making  a  bacterial  preparation. 

CONCAVE  SLIDES,  FERMENTATION  TUBES. 

The  concave  slide  is  ordinarily  used  for  making  hanging-drop  prepara- 
tions.     A  substitute  which  is  equally  good  may  be  made  by  spreading 


FIG.  3. — Hanging  drop,  over  hollow  ground  slide.     (Williams.} 

a  ring  or  square  of  vaselin — smaller  than  the  cover-glass  to  be  used — 
in  the  middle  of  the  slide.  Then  putting  a  loopful  of  salt  solution 
in  the  center  of  the  space,  and  inoculating  with  the  culture  to  be 
studied,  we  finally  cover  it  with  a  cover-glass,  gently  pressing  the  mar- 
gins down  on  the  vaselin.  This  gives  a  preparation  for  the  study 


10  APPARATUS. 

of  motility  or  agglutination  which  does  not  dry  out  for  hours,  and 
is  easier  to  focus  upon  than  the  concave  slide  hanging-drop  preparation. 
The  fermentation  tube  with  a  bulb  and  closed  arm  is  expensive, 
difficult  to  clean  and  is  easily  broken.  As  a  substitute  in  the  study 
of  gas  production  and  in  water  bacteriology,  the  Durham  tube  is  to  be 
recommended.  Into  a  test-tube,  about  i  x  7  in.,  we  introduce  the 
special  sugar  media,  then  drop  down  a  small  test-tube  (1/2  x  3  in.) 
with  its  open  end  downward.  Insert  the  plug  of  the  large  tube  and 
sterilize.  During  sterilization  the  fluid  enters  the  mouth  of  the  smaller 


FIG.  4. — Blood  serum  coagulating  apparatus. 

tube  and  fills  it,  and  when  the  medium  is  subsequently  inoculated,  if  gas 
forms,  it  appears  in  the  upper  part  of  the  closed  end  of  the  smaller  tube. 
For  inspissating  blood  serum  slants  a  regular  inspissator  is  desirable. 
This  is  nothing  more  than  a  double-walled  vessel,  the  space  between 
the  walls  being  filled  with  water.  As  a  substitute  one  may  take  the 
common  rice  cooker  (double  boiler).  Fill  the  outer  part  with  water; 
and  in  the  inner  compartment  pack  the  serum  tubes  properly  slanted 
on  a  piece  of  wood  or  a  wedge-shape  layer  of  cotton.  Place  a  weight 
on  the  cover  of  the  inner  compartment  to  sink  it  into  the  surrounding 
water,  and  allow  to  boil  for  one  or  two  hours.  This  same  apparatus 
may  be  used  for  their  sterilization  on  two  subsequent  days,  but  it  is 
better  to  sterilize  in  the  autoclave  or  Arnold.  As  regards  a  working 
desk,  it  will  be  found  convenient  to  have  an  arrangement  similar  to  the 
ordinary  flat-top  desk,  writh  a  tier  of  drawers  on  each  side.  A  block 


INCUBATORS.  H 

of  wood  with   holes  bored  in  it  to  contain  dropping-bottles  may  be 

placed  in  the  upper  left  hand  drawer.     In  this  way  the  stains  are  as 

accessible  as  if  they  encumbered  the 

desk.      It   is  advisable   to   paint   the 

inside  of  this  drawer  black  so  that  the 

light    may    not    cause    the    staining 

reagents  to  deteriorate. 

Ordinary  glass  salt  cellars  will  be 
found  very  useful,  where  the  watch- 
glass  is  employed.  They  may  also  be 

wrapped,  sterilized  and  used  to  con- 

.  FIG.  5. — Rice  cooker. 

tain  fluids  for  inoculating,  etc. 

For  use  in  making  loops  and  needles,  platinum  wire  of  26  gauge 
will  be  found  most  suitable.  The  handle  made  of  glass  rocNis  pref- 
erable to  the  metal  ones. 

INCUBATORS. 

\\hen  gas  is  obtainable,  the  maintaining  of  a  constant  temperature 
for  the  body  temperature  incubator  (38°  C.)  and  the  paraffin  oven 
(60°  C.)  is  best  secured  by  the  use  of  some  of  the  various  types  of 
thermo-regulators.  The  Reichert  type  is  the  one  in  general  use, 
although  there  are  many  features  about  the  Dunham  and  Roux 
regulators  which  are  advantageous. 

If  the  pressure  of  the  gas-supply  varies  from  time  to  time,  it  is 
essential  to  regulate  this  by  the  use  of  a  gas  pressure  regulator  (Murrill's 
is  a  cheap  and  satisfactory  one). 

Incubators,  controlled  electrically,  can  be  obtained  of  certain 
foreign  makers,  and  are  quoted  in  catalogues  of  American  dealers. 
It  is  probable  that  the  Koch  petroleum  lamp  incubator  is  the  most 
satisfactory  one  where  gas  is  not  obtainable.  They  should  be  of  all 
metal  construction,  and  not  with  a  wood  casing,  on  account  of  the 
danger  from  fire.  They  cost  from  twenty-five  to  fifty  dollars. 

An  incubator  may  be  extemporized  by  putting  the  bulb  of  an  incan- 
descent electric  lamp  in  a  vessel  of  water.  The  proper  temperature 
may  be  obtained  by  increasing  the  amount  of  water  or  by  covering  the 
opening  more  or  less  completely  with  a  towel.  The  test-tubes  to  be 


1 2  APPARATUS. 

incubated  can  be  put  into  a  fruit  jar  or  tin  can,  which  receptacle  is 
placed  in  the  vessel  heated  by  the  lamp. 

Emery  suggests  the  use  of  a  Thermos  bottle  as  an  incubator.  As 
regards  the  matter  of  a  low-temperature  incubator  (for  gelatin  work) , 
this  is  best  met. by  using  a  small  refrigerator.  The  ice  in  the  upper 
part  maintains  an  even  cold,  and  by  connecting  up  an  electric  lamp  in 
the  lower  part  of  the  refrigerator  we  can  easily  maintain  a  temperature 
which  only  varies  one  or  two  degrees  during  the  twenty-four  hours. 

With  a  i6-candle-power  lamp  a  temperature  of  about  25°  C.  is 
mantained  (this  is  too  high,  being  about  the  melting-point  of  gelatin), 
with  an  8  candle-power,  one  about  21°  to  23°  C.,  and  with  a  4-candle- 
power  from  18°  to  20°  C.;  the  box  being  about  20  x  30  x  36  inches. 

When  much  serum  reaction  work  is  done,  an  electrically  run  cen- 
trifuge is  of  the  greatest  convenience. 

A  filter  pump  attached  to  the  water  faucet,  preferably  by  screw 
threads,  is  almost  indispensable  for  filtering  cultures,'  etc.,  and  for 
cleaning  small  pipettes,  especially  the  hgemacytometer  pipettes.  Such  a 
filter  or  vacuum  pump  with  a  vacuum  gauge  is  more  easily  controlled. 

BACTERIOLOGICAL  PIPETTES. 

With  the  possible  exception  of  the  platinum  loop,  there  is  no  piece 
of  apparatus  so  applicable  to  many  uses  as  the  capillary  pipette  made 
from  a  piece  of  glass  tubing. 

These  may  be  made  in  a  great  variety  of  shapes.  The  one  with  a 
hooked  end,  the  Wright  tube,  is  the  best  apparatus  for  securing  blood 
for  serum  tests.  The  crook  hangs  on  the  centrifuge  guard  and  by 
filing  and  breaking  the  thicker  part  of  the  tube  the  serum  is  accessible 
to  a  capillary  rubber  bulb  pipette  or  to  the  tip  of  a  haemacytometer 
pipette.  In  this  way  dilutions  of  serum  are  easily  made.  The  capil- 
lary pipette  is  made  by  taking  a  piece  of  1/4  in.  soft  German  glass 
tubing,  about  six  inches  long,  and  heating  in  the  middle  in  a  Bunsen 
flame,  revolving  the  tubing  while  heating  it.  When  it  becomes  soft 
in  the  center,  remove  from  the  flame  and  with  a  steady  even  pull 
separate  the  two  ends.  The  capillary  portion  should  be  from  eighteen 
to  twenty  inches  in  length.  When  cool,  file  and  break  off  this  capillary 
portion  in  the  middle.  We  then  have  two  capillary  pipettes.  By 


BACTERIOLOGICAL    PIPETTES. 


using  a  rubber  bulb,  such  as  comes  on  medicine  droppers,  \ve  have  a 
means  of  sucking  up  and  forcing  out  fluids  by  pressure  with  the  thumb 
and  forefinger  of  the  right  hand.  The  bulb  should  be  pushed  on  about 
1/2  to  3/4  in.;  this  gives  a  firmer  surface  to  control  the  pressure  on 
the  bulb. 


FIG.  6. — i,  2,  3,  Drawing  out  glass  tubing;  4,  5,  Wright's  rubber  bulb  capillary 
pipettes  showing  grease  pencil  mark  for  making  dilutions;  6,  7,  Wright's  U  tubes; 
8,  9,  10,  Methods  of  drawing  out  test  tubes  for  vaccines  in  opsonic  work;  n,  Bac- 
teriological pipette. 

A  bacteriological  pipette  is  made  by  drawing  out  a  nine-inch  piece 
of  tubing  about  three  inches  at  either  end,  then  heating  in  the  middle 
we  draw  out  and  have  two  pipettes  similar  to  the  one  shown  in  the 
drawing.  A  piece  of  cotton  is  loosely  pushed  in  just  above  the  narrow 
portion.  These  may  be  wrapped  in  paper  and  sterilized  for  future1 
use.  They  may  be  made  perfectly  sterile  at  the  time  of  drawing  out. 

Where  gas  is  not  at  hand,  the  Barthel  alcohol  lamp  gives  a  flame 
similar  to  that  of  the  Bunsen  lamp  and  is  equally  satisfactory  for 
heating  glass  tubing. 


CHAPTER  II. 
CULTURE  MEDIA. 

WHILE  there  are  certain  advantages  in  sterilizing  the  glass  test- 
tubes  prior  to  filling  them  with  media,  yet  this  may  be  dispensed  with — 
the  sterilization  after  the  media  has  been  tubed  being  sufficient.  If  a 
dressing  sterilizer  is  at  hand,  this  is  preferable  for  sterilizing  such 
media  as  bouillon,  potato  and  agar  (10  to  15  pounds'  pressure  for 
fifteen  .minutes) .  Milk  shculd  be  sterilized  with  the  Arnold,  subjecting 
the  media  to  three  steamings  for  twenty  minutes  on  three  successive 
days.  Gelatin  may  be  sterilized  in  either  way,  but  preferably  in  the 
autoclave  at  7  pounds'  pressure  for  fifteen  minutes.  As  soon  as  taken 
out  of  the  sterilizer  it  should  be  cooled  as  quickly  as  possible  in  cool 
water. .  This  procedure  tends  to  prevent  the  lowering  of  the  melting- 
point  of  the  finished  gelatin  and  also  preserves  its  spissitude. 

Blood-serum  is  preferably  solidified  as  slants  in  a  blood  serum 
inspissator.  This  requires  one  to  two  hours.  The  subsequent  sterili- 
zation in  the  autoclave  or  Arnold  should  not  be  done  immediately 
after  making  the  solidified  slants,  but  on  the  subsequent  day.  If 
done  on  the  same  day,  many  of  the  slants  are  ruined  by  being  dis- 
rupted by  bubbles.  The  preparation  of  blood-serum  slants  or  slants 
of  egg  media  can  be  conveniently  carried  out  in  a  rice  cooker  (double 
boiler).  Place  the  tubes  in  the  inner  compartment  of  the  cooker, 
obtaining  the  slant  desired  by  manipulating  an  empty  test  tube,  or 
with  a  towel  or  cotton  batting  on  the  bottom.  Then  cover  the  tubes 
with  another  towel.  The  outer  compartment  should  contain  water 
alone '(no  25%  salt  solution).  The  inner  compartment  should  be 
weighied  down  so  that  it  is  surrounded  by  water — the  light  tubes  not 
being  sufficient  to  sink  it.  Allowing  the  water  in  the  outer  compart- 
ment to  boil  one  or  two  hours  will  inspissate  or  solidify  the  slants 
satisfactorily.  The  sterilization  on  subsequent  days  may  be  carried 
out  in  the  same  apparatus,  although  it  is  more  efficient  if  done  in  an 

14 


M  TRIKM     1501  II  i  15 

Arnold  or  an  autoclave.  (This  sterilization  in  the  rice  cooker  makes 
the  media  too  dry.) 

In  making  media  a  rice  cooker  is  almost  essential;  at  any  rate,  it  is 
so  if  ease,  expedition  and  unfailing  success  in  preparation  are  to  be 
achieved.  As  it  is  necessary  to  make  the  contents  of  the  inner  com- 
partment boil,  the  temperature  of  the  water  in  the  outer  compartment 
must  be  raised.  This  is  done  by  using  a  25%  solution  of  common  salt 
or  a  20%  solution  of  calcium  chloride  in  the  outer  compartment  instead 
of  plain  water. 

A  15%  solution  of  salt  raises  the  boiling  point  2  1/2°  C.;  a  20%, 
3  1/2°  C.,  and  a  25%,  4  1/2°  C.  The  raising  of  the  boiling- 
point  by  calcium  chloride  is  about  the  same  for  similar  strength 
solutions. 

Although  the  Bacteriological  Committee  of  the  A.  P.  H.  Asso- 
ciation recommends  special  steps  to  be  taken  in  the  preparation  of 
gelatin  and  agar,  yet  for  clinical  purposes  it  will  be  found  satisfactory 
to  keep  on  hand  a  stock  of  bouillon,  and  when  it  is  desired  to  make 
agar  or  gelatin  to  simply  prepare  such  media  from  the  stock  bouillon 
in  the  way  to  be  subsequently  given. 

XUTRIEXT  BOUILLON. 

This  may  be  made  either  from  fresh  meat  or  from  meat  extract. 
Media  from  fresh  meat  are  usually  lighter  in  color  and  possibly  clearer. 
In  the  Philippines,  however,  certain  measures  employed  for  the  preser- 
vation of  the  meat  made  it  very  difficult  to  prepare  clear  bouillon  from 
it,  so  that  meat  extract  was  used  entirely.  There  is  very  little  differ- 
ence, if  any,  in  the  nutritive  power  of  media  made  in  either  way. 
The  chief  objections  to  fresh  meat  as  a  base  are:  (i)  It  takes  more 
time  and  trouble.  (2)  The  reaction,  due  to  sarcolactic  acid  and  acid 
salts,  is  quite  acid,  so  that  it  is  necessary  to  titrate  and  neutralize  the 
excess  of  acidity.  (3)  The  reaction  of  the  finished  media  tends  to 
change  unless  the  boiling  at  the  time  of  making  was  very  prolonged. 
(4)  It  is  not  infrequent  to  have  a  heavy  precipitate  of  phosphates 
thrown  down  at  the  time  of  sterilization,  thus  making  it  necessarv  to 
repeat  the  process  of  filtration  and  sterilization. 


1 6  CULTURE    MEDIA. 

If  fresh  meat  is  used,  take  about  500  grams  (one  pound),  remove 
fat  and  cut  it  up  with  a  sausage  mill  or  purchase  the  meat  already 
cut  up  as  fora  Hamburg  steak.  It  makes  little  difference  whether  the 
amount  be  100  grams  more  or  less.  Place  the  chopped-up  meat  in  a 
receptacle  and  pour  1000  c.c.  of  water  over  it.  Keep  in  the  ice  chest 
over  night  and  the  next  morning  skim  off  with  a  piece  of  absorbent 
cotton  the  scum  of  fat;  then  squeeze  out  the  infusion  with  a  strong 
muslin  cloth,  making  the  amount  up  to  1000  c.c.  This  meat 
infusion  contains  all  the  albuminous  material  necessary  for  the 
clarification  of  the  bouillon.  It  is  convenient  to  designate  this 
meat  base  as  Meat  Infusion  to  distinguish  from  the  base  containing 
meat  extract. 

Having  obtained  1000  c.c.  of  this  50%  meat  infusion,  we  dissolve  in 
it  i%  of  Witte's  peptone  and  1/2%  of  sodium  chloride.  While  there  is 
a  sufficiency  of  the  various  salts  necessary  for  bacterial  development 
in  the  meat  juices,  yet  there  is  not  enough  to  give  the  best  results  when 
bouillon  cultures  of  various  organisms  are  used  for  agglutination  tests; 
and  futhermore,  when  bouillon  is  used  for  blood  cultures,  disinte- 
gration of  the  red  cells,  with  clouding  of  the  clear  medium,  may  occur 
if  there  be  not  sufficient  salt  present  to  prevent  this. 

The  salt  and  the  peptone  are  best  put  in  a  mortar,  and  adding  about 
one  ounce  of  the  meat  infusion  we  make  a  pasty  mass;  then  we  grad- 
ually add  the  remaining  infusion  until  solution  is  complete.  It  is 
sometimes  recommended  to  use  a  temperature  of  50°  C.  to  facilitate 
the  solution  of  the  peptone.  This  is  not  necessary,  and  if  the  tempera- 
tare  is  not  watched  closely  it  might  go  up  to  65°  C.  or  higher  and  we 
should  lose  the  clearing  albuminous  material  from  its  coagulation. 
Of  this  rather  cloudy  solution  take  up  10  c.c.  with  a  pipette  and  let  it 
run  out  into  a  beaker.  Add  forty  c.c.  of  distilled  or  rain  water  and 
about  six  drops  of  a  0.5%  phenolphthalein  solution.  (Phenolphtha- 
lein,  0.5;  dilute  alcohol,  100  c.c.)  Now  from  a  burette  filled  with 
decinormal  sodium  hydrate  solution,  run  in  this  solution  until  we  have 
the  development  of  a  rich  violet-pink  color  in  the  diluted  bouillon  in  the 
beaker.  To  obtain  a  standard  for  comparison,  simply  add  six  drops  of 
the  phenolphthalein  solution  to  50  c.c.  of  water  and  add  about  i  c.c.  of 
the  N/io  sodium  hydrate  solution.  As  soon  as  we  have  obtained  a 


TITRATION   OF   MEDIA.  IJ 

color  of  the  same  intensity  as  our  standard,  we  read  off  the  number  of 
c.c.  or  fractions  of  a  c.c.  of  N/io  sodium  hydrate  solution  added  to 
produce  the  color.  This  number  gives  the  acidity  of  the  bouillon  in 
percentage  of  N/i  acid  solution.* 

Percent  acid  means  that  so  many  c.c.  of  N/i  acid  added  to  100  c.c. 
of  the  medium  at  the  neutral  point  would  give  that  percentage  reaction. 
Thus  i  1/2  c.c.  of  N/i  HC1  solution  added  to  100  c.c.  of  medium  at  o, 
would  give  us  i  1/2%  of  acidity  or  +1.5. 

Percent  alkaline  means  so  many  c.c.  of  N/i  sodium  hydrate 
solution  added  to  100  c.c.  of  the  medium  at  the  neutral  point.  Thus 
a  1/2%  alkaline  medium  would  be  one  whose  alkalinity  would  cor- 
respond to  the  addition  of  1/2  c.c.  of  N/i  NaOH  to  100  c.c.  of  the 
medium  at  o.  It  is  written  — .5. 

If  we  took  100  c.c.  of  the  medium  and  put  it  in  a  beaker  and  then 
ran  in  N/i  NaOH  solution  from  a  burette,  it  will  be  readily  under- 
stood that  if  we  had  to  add  31/2  c.c.  of  N/ 1  NaOH  to  obtain  the  pink 
color,  it  would  show  that  the  acidity  of  the  100  c.c.  of  medium,  being 
tested,  corresponded  to  3.5  c.c.  of  N/i  acid  solution,  and  that  its 
acidity  was  equal  to  3  1/2%  of  N/i  acid  solution,  or  that  its  reaction 
was  +3.5. 

As  N/ 1  NaOH  solution  is  too  corrosive  for  general  use  in  a  burette, 
and  as  10  c.c.  of  medium  is  more  convenient  to  work  with  than  100  c.c., 
we  use  a  solution  one-tenth  the  strength  of  the  N/i  NaOH  and  we  take 
only  one-tenth  of  the  100  c.c.  of  medium.  In  this  way  it  is  the  same 
from  a  stand-point  of  directly  reading  off  our  percentage  reaction  as  if 
we  had  100  c.c.  of  medium  and  used  N/i  NaOH  solution.  The 
A.  P.  H.  Association  recommends  5  c.c.  of  the  medium  and  the  use  of 
X/20  NaOH.  As  the  N/io  NaOH  is  always  at  hand  for  titrating 
gastric  juice,  the  N/io  is  used  instead. 

*  A  more  satisfactory  method  for  one  with  experience  in  titrating  is  to  continue 
to  add  the  N/io  XaOH  solution  from  the  burette,  drop  by  drop,  until  the  addition 
of  a  drop  fails  to  show  any  intensifying  of  the  purplish  violet  color  at  the  spot 
where  it  came  in  contact  with  the  diluted  bouillon  in  the  beaker.  This  marks  the 
end  reaction.  A  reaction  of  about  +.7  in  the  cold  gives  a  delicate  pink.  In  titrating 
at  the  boiling  temperature  (probably  preferable  for  gelatine  and  agar)  use  a  por- 
celain dish. and  boil  for  i  or  2  minutes.  Then  add  the  sodium  hydrate  solution 
from  the  burette  until  a  faint  pink  develops.  Then  add  about  4  additional  drops 
from  the  burette  and  the  reading  will  be  fairly  accurate. 


l8  CULTURE    MEDIA. 

Having  determined  the  percentage  acidity  of  the  10  c.c.  sample 
tested,  we  easily  calculate  the  number  of  c.c.  of  N/iNaOH  solution 
required  to  be  added  to  the  1000  c.c.  of  bouillon  to  obtain  a  reaction 
corresponding  to  the  neutral  point  of  phenolphthalein.  It  is  more  exact 
to  take  the  average  of  two  titrations. 

As  100  c.c.  of  medium  would  require  31/2  c.c.,  1000  c.c.  would 
require  10  times  as  much,  or  35  c.c.  N/i  NaOH  solution.  Having 
measured  out  and  added  35  c.c.  of  the  N/i  NaOH  solution  to  the 
meat  infusion,  containing  salt  and  peptone,  we  have  a  solution  which  is 
exactly  neutral  to  phenolphthalein,  or  o.  It  is  usually  considered  that 
a  reaction  of  about  i  percent  acid  is  the  optimum  reaction  for  bacterial 
growth.  Hence  we  should  now  add  i  %  of  N/i  HC1  solution  to  the 
medium.  This  would  be  accomplished  by  adding  10  c.c.  of  N/i 
HC1  solution  to  the  1000  c.c.  of  neutralized  medium,  and  we  would 
have  a  medium  with  a  reaction  of  + 1.  If  we  desired  a  reaction  of  one 
percent  alkalinity  we  would  add  an  additional  c.c.  of  N/i  NaOH 
solution  to  every  100  c.c.  of  the  medium  at  o,  or  10  c.c.  for  the  1000 
c.c.  of  medium.  The  reaction  would  then  be  — i. 

As  a  matter  of  convenience,  we  usually  determine  the  reaction  of  the 
medium,  which  is  always  more  or  less  acid,  and  then  add  enough  N/i 
NaOH  to  reduce  the  acidity  to  the  percentage  we  desire  to  set  the 
medium,  instead  of  neutralizing  all  the  acidity  present  and  then,  in  a 
second  operation,  restoring  the  acidity  to  the  point  desired. 

Thus  finding  the  acidity  of  the  medium  to  be  3  1/2%  and  desiring  to 
give  it  an  acidity  of  i%,  we  would  add  only  21/2  c.c.  of  N/i  NaOH 
to  every  100  c.c.  of  medium,  or  25  c.c.  for  the  1000  c.c.  of  medium. 
The  reaction  would  then  be  found  to  be  -fi. 

The  neutral  point  of  litmus  is  not  a  sharp  one,  but  it  corresponds 
rather  closely  with  a  reaction  of  +1.5  to  phenolphthalein.  The 
recommendations  of  the  A.  P.  H.  Association  call  for  making  the 
titration  with  the  medium  boiling.  This  is  a  very  difficult  titration 
and  students  obtain  results  varying  greatly,  which  is  not  the  case  when 
the  titration  is  conducted  at  room  temperature  and  a  standard  color  is 
at  hand.  If  the  color  of  the  end  reaction  at  boiling-point  be  obtained, 
it  will  be  found  that  when  cool  it  deepens  until  it  corresponds  to  the 
rich  violet-pink  of  the  end  reaction  in  the  cold  or  vice  versa. 


i  1 1. 1. o.\.  ig 

To  summarize: 

Take  Peptone,  10  grams 

Sodium  chloride,  5  grams 

50%  meat  infusion,  1000  c.c. 

Dissolve  the  peptone  and  sodium  chloride  in  the  meat  infusion  and 
add  enough  N/i  NaOH  to  make  the  reaction  +i. 

Put  the  solution  in  the  inner  compartment  of  a  rice  cooker  and  bring 
to  the  boiling-point  and  maintain  this  temperature  for  twenty  minutes. 
The  calcium  chloride  or  sodium  chloride  in  the  outer  compartment  of 
the  rice  cooker  enables  us  to  secure  a  boiling  temperature  for  the  con- 
tents of  the  inner  compartment.  Do  not  stir  the  bouillon  that  is  being 
heated,  as  the  pultaceous  membranous  mass  of  coagulated  albumin 
makes  filtration  easy.  Filter.  The  filter-paper  in  the  funnel  should  be 
thoroughly  wet  with  water  before  pouring  on  the  bouillon.  This  is  to 
prevent  clogging  of  the  pores  of  the  filter-paper.  Make  up  the  quantity 
of  filtrate  to  1000  c.c.  by  adding  water. 

If  greater  exactness  is  demanded  than  answers  for  ordinary 
clinical  work,  it  is  advisable  to  again  titrate  and  again  adjust  the 
reaction  or  to  simply  record  the  exact  reaction.  It  is  more  convenient 
to  have  a  counterpoise  to  balance  the  inner  compartment  and  then  to 
add  water  to  the  medium  until  a  kilo  weight,  in  addition  to  the  weight 
balancing  the  container,  is  just  balanced,.  Then  titrate,  adjust  the 
reaction  (if  so  desired)  and  filter.  Sterilize  in  the  autoclave  at  115° 
to  120°  C.  for  fifteen  minutes  or  in  the  Arnold  on  three  successive  days. 
The  use  of  a  balance  is  preferable  in  the  preparation  of  bouillon, 
necessary  in  making  gelatin  and  imperative  in  making  agar  media 

BOUILLON  MADE  FROM  LIEBIG'S  MEAT  EXTRACT. 

Place  in  a  mortar  3  grams  of  Liebig's  extract,  10  grams  of  pep- 
tone and  5  grams  of  sodium  chloride.  Dissolve  the  whites  of  one  or 
two  eggs  in  1000  c.c.  of  water.  Then  add  this  egg-white  water,  little 
by  little,  to  the  extract,  peptone  and  salt  in  the  mortar  until  a  brownish 
solution  is  obtained.  Pour  this  into  the  inner  compartment  of  a  rice 
cooker;  apply  heat  to  the  outer  compartment  containing  the  salt  or 
calcium  chloride  solution,  allow  to  come  to  a  boil  and  to  continue 


20  CULTURE    MEDIA. 

boiling  for  fifteen  to  twenty  minutes.  Do  not  stir.  Place  inner  com- 
partment on  the  scales  and  its  counterpoise  and  a  one-kilo  weight  on 
the  other  side.  Add  water  until  the  two  arms  balance.  Filter  and 
sterilize. 

The  reaction  of  media  made  with  Liebig's  meat  extract  rarely 
exceeds  +.75  (from  +.6  to  +.9).  Consequently  for  growing  bacteria 
it  is  unnecessary  to  titrate  and  adjust  reaction  unless  precision  is 
demanded. 

SUGAR-FREE  BOUILLON. 

Inoculate' nutrient  bouillon  in  a  flask  with  the  colon  bacillus. 
Allow  to  incubate  at  37°  C.  over  night.  Pour  the  contents  into  a  sauce- 
pan and  bring  to  a  boil  to  kill  the  colon  bacilli.  Put  about  15  grams 
of  purified  talc  (Talcum  purificatum,  U.  S.  P.)  in  a  mortar.  Add  the 
dead  colon  culture,  stirring  constantly.  Then  filter  through  filter- 
paper.  It  may  be  necessary  to  again  pass  the  filtrate  through  the 
same  filter  until  the  sugar-free  bouillon  is  perfectly  clear. 

For  all  ordinary  purposes  the  very  small  amount  of  sugar  in  bouillon 
made  from  Liebig's  meat  extract  may  be  neglected  in  determining  gas 
production;  so  that  under  such  conditions  the  various  sugars  could  be 
added  directly  to  the  meat-extract  bouillon. 

SUGAR  BOUILLONS. 

The  sugar  media  ordinarily  used  for  determining  fermentation 
or  gas  production  are  those  of  glucose  and  lactose.  In  special  work 
such  carbohydrates  as  saccharose  and  maltose  are  used.  The  alcohol 
mannite  is  used  in  differentiating  strains  of  dysentery  bacilli. 

To  make,  simply  dissolve  i  or  2%  of  the  sugar  in  sugar-free  bouillon 
or  that  made  from  meat  extract.  Tube  in  Durham's  or  the  ordinary 
fermentation  tubes  and  sterilize  in  the  autoclave  at  only  about  5 
pounds'  pressure  for  15  minutes,  or  in  the  Arnold. 

GLYCERIN  BOUILLON. 

Add  6%  of  glycerin  to  ordinary  bouillon.  It  is  chiefly  used  in  the 
cultivation  of  tubercle  bacilli. 


MTRIENT  AGAR.  21 

PEPTONE  SOLUTION  (DUNHAM'S). 

Dissolve  i'  ,  of  Witte's  peptone  and  1/2%  of  sodium  chloride  in 
distilled  water.  Filter,  tube  and  sterilize.  Peptone  solution  may  he 
used  as  a  base  for  sugar  media  instead  of  bouillon.  It  is  the  medium 
used  in  testing  for  indol  production.  This  test  is  made  by  adding 
from  six  to  eight  drops  of  concentrated  H2SO4  to  a  24-  to  48-hour-old 
peptone  culture  of  the  organism  to  be  tested.  If  the  organism  pro- 
duces both  indol  and  a  nitroso  body,  we  obtain  a  violet-pink  coloration, 
"cholera  red."  If  no  pink  color  is  produced  on  the  addition  of  the 
sulphuric  acid,  add  about  i  c.c.  of  an  exceedingly  dilute  solution 
(i  :  10,000)  of  sodium  nitrite. 

XITRIEXT  AGAR. 

In  making  agar  medium  it  is  preferable  to  use  powdered  agar,  as 
this  goes  into  solution  more  readily  than  the  shredded  agar.  The 
reaction  of  agar  is  slightly  alkaline,  so  that  if  i  1/2  to  2%  of  agar  is 
added  to  nutrient  bouillon  having  a  reaction  of  + 1  the  finished  prod- 
uct will  be  found  to  be  about  -f  .8. 

To  make:  Weigh  15  to  20  grams  of  powdered  agar  and  place  in  a 
mortar.  Make  a  paste  by  adding  nutrient  bouillon,  little  by  little, 
and  when  a  smooth  even  mixture  is  made,  pour  it  into  the  inner  com- 
partment of  a  rice  cooker  and  add  the  remainder  of  the  1000  c.c.  of 
bouillon.  The  use  of  the  balance  is  preferable. 

The  outer  compartment  of  the  rice  cooker  should  contain  the  25% 
salt  solution.  Bring  to  boil,  and  the  agar  will  be  found  to  have 
entirely  gone  into  solution  after  three  to  five  minutes  of  boiling. 

Then,  using  a  funnel  which  has  been  heated  in  boiling  water  and 
which  contains  a  small  pledget  of  absorbent  cotton,  we  filter  the  agar, 
tube  it  and  sterilize  it  in  the  autoclave  or  Arnold.  One  and  one-half 
percent  agar  can  be  readily  filtered  through  filter-paper  and  gives  a 
clearer  medium. 

By  taking  of  meat  extract  3  grams,  peptone  10  grams,  salt  5  grams, 
powdered  agar  15  grams,  the  white  of  one  egg  and  1000  c.c.  of  water, 
making  at  first  a  paste  of  all  the  ingredients  in  a  mortar,  then  gradually 
adding  the  remainder  of  the  1000  c.c.  of  water,  putting  in  the  rice 


22  CULTURE    MEDIA. 

cooker,  bringing  to  a  boil  without  stirring,  allowing  to  boil  fifteen 
minutes  and  then  filtering  through  absorbent  cotton  in  a  hot  funnel, 
we  obtain  a  satisfactory  medium,  the  reaction  of  which  will  be  from 
+  .7  to  +.9. 

GLUCOSE  AGAR. 

Add  the  agar  to  i  or  2%  glucose  bouillon  and  proceed  as  for  ordinary 
agar. 

GLYCERIN  AGAR. 
Add  the  agar  to  6%  glycerin  bouillon  instead  of  nutrient  bouillon. 

GLYCERIN  AGAR  EGG  MEDIUM. 

Take  the  white  and  the  yolk  of  one  egg  and  mix  thoroughly  in  a 
vessel  kept  between  45°  and  55°  C.  with  an  equal  amount  of  glycerin 
agar.  Tube  the  medium,  inspissate  in  a  rice  cooker  as  for  serum 
tubes,  and  sterilize  as  for  blood-serum  tubes. 

This  makes  an  excellent  medium  for  growing  tubercle  bacilli.  As 
egg  medium  has  a  tendency  to  be  dry,  it  is  well  to  add  i  c.c.  of  glycerin 
bouillon  to  each  slant  before  autoclaving. 

NUTRIENT  GELATIN. 

Add  about  12%  (120  grams)  of  "gold  label"  gelatin  to  1000  c.c. 
of  nutrient  bouillon  in  a  rice  cooker.  If  the  bouillon  had  a  reaction  of 
about  +i,  the  gelatin  solution  will  be  about  +3.5,  so  that  it  is  always 
necessary  to  titrate  gelatin  and  neutralize  to  about  +i.  The  pro- 
cedure is  the  same  as  for  bouillon.  As  the  color  reaction  is  not  quite 
as  distinct  with  gelatin,  it  is  better  to  make  a  color  standard  with  4 
or  5  c.c.  of  the  gelatin  medium  in  50  c.c.  of  water,  instead  of  using 
the  distilled  water  alone,  as  was  recommended  for  bouillon. 

Having  neutralized  and  allowed  to  boil  for  fifteen  minutes,  we  filter 
through  filter-paper  in  a  hot  funnel.  As  it  is  very  important  that 
gelatin  should  be  perfectly  clear,  it  is  better  to  filter  through  filter- 
paper  than  through  cotton.  The  filter  paper  should  be  very  thoroughly 
wetted  with  very  hot  water  before  filtering  gelatine  or  agar. 

Tube  the  medium  and  sterilize,  either  in  the  Arnold  on  three 


POTATO    Ml.DIA. 


successive  days  or  in  the  autoclave  at  8-10  pounds'  pressure  for  ten 
minutes.  The  tubes  should  be  cooled  as  quickly  as  possible  in  cold 
water  after  taking  out  of  the  sterilizer. 

LITMUS  MILK. 

Milk  for  media  should  be  as  fresh  as  possible.  It  should  then  be 
put  in  a  1000  c.c.  Erlenmeyer  flask,  sterilized  for  fifteen  minutes  in  the 
Arnold  and  set  over  night  in  the  refrigerator.  The  next 
morning  the  milk  beneath  the  cream  should  be  siphoned 
off.  The  short  arm  of  the  siphon  should  not  reach  the 
bottom  of  the  flask  so  as  to  avoid  the  sediment.  Add 
sufficient  tincture  of  litmus  to  this  milk  to  give  a  decided 
lilac  tinge;  tube  and  sterilize  in  the  Arnold  on  three  suc- 
cessive days. 

POTATO  SLANTS. 

Take  Irish  potatoes  and  scrub  thoroughly  with  a  stiff 
brush.  Then  pare  off  generously  all  the  outer  portion. 
From  the  white  interior  cut  out  cylinders  with  a  cork 
borer.  These  cylinders  should  be  of  1/2  to  3/4  of  an 
inch  in  diameter.  Divide  a  cylinder  by  a  diagonal  cut. 
This  gives  a  plug  with  a  flat  base,  the  other  extremity 
being  a  slant.  These  potato  plugs  should  be  left  in 
running  water  over  night  or  washed  with  frequent  changes 
of  water.  This  prevents  the  blackening  of  the  plug. 
Into  a  i -in.  test-tube  drop  a  pledget  of  absorbent  cotton 
well  moistened  with  water.  Then  drop  in  the  potato  plug,  FlG 
base  downward.  Sterilize  in  the  autoclave  at  15  pounds  Potato  in 
for  fifteen  to  twenty  minutes,  to  insure  sterility. 

For  glycerin  potato,  soak  the  plugs  in  6%  glycerin 
solution  for  about  one  hour.  Then  drop  in  a  pledget  of  absorbent 
cotton  moistened  with  the  same  glycerin  solution  into  the  test-tubes 
and  follow  it  with  the  potato  plug.  Sterilize  in  the  autoclave. 

BLOOD-SERUM. 

The  blood  of  cattle  should  be  collected  in  large  pans  or  pails  at  the 
abattoir.     This  vessel  of  blocd  should  then  be  kept  in  the  cold-storage 


24  CULTURE    MEDIA. 

room  and  the  next  morning  the  more  or  less  clear  serum  will  have  been 
squeezed  out  from  the  clot.  Collect  this  serum  and  keep  in  the  ice 
chest  for  future  use.  If  to  be  kept  for  a  long  time,  it  is  advisable  to  add 
about  2%  of  chloroform  to  the  serum.  This  will  not  only  keep  the 
serum,  but  will  eventually  sterilize  it. 

To  make  Loffler's  serum,  take  one  part  of  glucose  bouillon  and 
three  parts  of  blood-serum.  Mix,  tube  and  coagulate  the  albumin  in 
the  inspissator  or  rice  cooker,  giving  the  tubes  a  proper  slant  before 
heating.  Sterilize  the  following  day  in  the  autoclave  as  previously 
directed  or  in  the  Arnold  on  three  successive  days. 

A  SUBSTITUTE  FOR  ORDINARY  BLOOD -SERUM. 

Purchase  a  good  article  of  commercial  blood-serum  albumin  and 
make  a  15%  solution  of  it  in  glucose  bouillon.  Tube  and  inspissate  as 
for  blood-serum.  If  made  with  glycerin  bouillon,  it  makes  a  good 
medium  for  tubercle  bacilli. 

As  this  dried  blood  albumin  only  costs  about  fifty  cents  a  pound 
and  will  keep  permanently,  it  is  exceedingly  convenient  for  those  not 
near  an  abattoir.  Its  use  was  first  suggested  by  Hospital  Steward  King, 
of  this  laboratory,  and  I  cannot  find  that  it  has  been  previously  used 
as  a  substitute  for  fresh  blood-serum. 

At  any  rate,  the  results  with  it  as  a  culture  medium  seem  to  be  the 
same  as  with  the  fresh  serum. 

This  is  better  than  the  various  white  of  egg  substitutes  usually 
recommended. 

HYDROCELE,  AND  BLOOD  AGAR. 

To  tubes  of  melted  agar  at  50°  C.  add  from  one  to  three  c.c.  of 
hydrocele  or  ascitic  fluid,  observing  aseptic  precautions.  For  blood 
agar  the  blood  from  a  vein  should  be  received  into  a  sodium  citrate  salt 
solution  to  prevent  coagulation,  and  added  subsequently  as  for  hydro- 
cele fluid.  Allow  the  agar  to  solidify  as  a  slant. 

BLOOD-STREAKED  AGAR. 

Sterilize  the  lobe  of  the  ear  and  puncture  with  a  sterile  needle. 
Collect  the  exuding  blood  on  a  large  platinum  loop  and  smear  it  over 


F.ECES    PLATING    MEDIA.  25 

the  surface  of  an  agar  slant.     It  is  advisable  to  incubate  overnight  as  a 
test  for  sterility. 

BILE  MEDIA. 

Secure  ox  bile  from  the  abattoir  or  human  bile  from  cases  of  gall- 
bladder drainage  in  hospitals.  Put  about  10  c.c.  in  each  tube  and 
sterilize.  Some  prefer  to  add  i%  of  peptone. 

This  is  the  medium  for  blood  cultures  in  typhoid,  etc. 

PLATING  MEDIA  FOR  RECES  WORK. 

The  media  of  Endo,  Conradi-Drigalski  and  the  lactose  litmus  agar 
medium  are  probably  the  most  satisfactory  of  the  numerous  ones  that 
have  been  proposed  for  plating  out  faeces.  A  convenient  way  of  pre- 
paring any  one  or  all  of  these,  and  which  apparently  gives  media  equal 
to  that  prepared  according  to  the  original  formulae,  is  as  follows: 

Liebig's  extract,  5  grams. 

Salt,  5  grams. 

Peptone,  10  grams. 

Lactose  (C.  P.),  10  grams. 

Agar,  20  grams. 

Water  to  make,  1000  c.c. 

Prepare  as  for  ordinary  nutrient  agar,  with  the  difference  that 
the  reaction  should  be  brought  down  to  — .5. 

For  Endows  Medium. — Keep  this  lactose  agar  base  in  100  c.c. 
quantities  in  Erlenmeyer  flasks  instead  of  test-tubes. 

When  needed  for  plating,  melt  a  flask  of  this  agar,  and  while  liquid 
add  to  the  100  c.c.  ten  drops  of  a  saturated  alcoholic  solution  of  basic 
fuchsin,  and  then  twenty  drops  of  a  freshly  prepared  20%  solution  of 
sodium  sulphite.  The  medium  should  be  of  a  light  flesh  or  pale 
salmon  color. 

Colon  bacilli  show  on  this  medium  as  vermilion  colonies,  which  in 
about  48  hours  have  a  metallic  scum  on  them.  Typhoid  and  dysentery 
colonies  are  grayish. 

For  Lactose  Litmus  Agar. — Color  the  lactose  agar  base  with 
tincture  of  litmus  to  a  lilac  color.  This  may  be  tubed,  putting  10  c.c. 


26  CULTURE    MEDIA. 

in  each  test-tube,  or  put  in  quantities  of  50  or  100  c.c.  in  small  Erlen- 
meyer  flasks.  It  is  then  sterilized  in  the  autoclave  (10  pounds  for  15 
minutes)  or  in  the  Arnold. 

For  Conradi-Drigalski  Medium. — Melt  down  the  lactose  litmus 
agar  in  the  flasks  holding  100  c.c.  Then  add  to  the  100  c.c.  of  medium, 
i  c.c.  of  a  solution  of  crystal  violet  (crystal  violet  o.i  gram,  distilled  water 
100  c.c.).  The  medium  is  then  ready  to  put  into  plates.  Colon 
colonies  are  pink.  Typhoid  and  dysentery  colonies,  a  bluish-gray. 


CHAPTER  III. 
STAINING  METHODS. 

L\  order  to  study  a  bacterial  or  blood  specimen  the  first  essential  is 
a  properly  prepared  film  the  matter  of  staining  is  of  less  importance. 
The  slide  or  cover  glass,  after  cleaning  with  soap  and  water  or  by 
special  solutions,  should  be  polished  with  a  piece  of  old  linen.  If  a 
glass  surface  is  free  of  grease  a  loopful  of  water  will  smear  out  evenly 
and  over  the  entire  surface.  The  only  quick  practical  way  to  make 
the  slide  or  cover -glass  grease  free  is  to  burn  the  surface  for  a  moment 
in  a  Bunsen  or  alcohol  flame.  The  cover -glass  must  not  be  warped. 
To  make  a  preparation,  apply  a  small  loopful-of  distilled  water  on  the 
slide  or  cover  glass  and,  touching  a  colony  with  a  platinum  needle, 
stir  the  transferred  culture  into  the  loopful  (not  drop)  of  water.  The 
mistake  is  almost  invariably  made  of  taking  up  too  much  bacterial 
growth.  Fluid  cultures  do  not  need  dilution.  Smearing  the  mixture 
over  a  large  part  of  the  cover-glass  or  over  an  equal  area  of  a  slide,  it  is 
allowed  to  dry.  If  very  little  water  is  used,  the  preparation  dries 
readily.  Otherwise  it  can  be  dried  in  the  fingers  high  over  a  flame. 
As  soon  as  dry,  the  cover  glass  should  be  passed  three  times  through 
the  flame,  film  side  up,  to  fix  the  preparation.  Slides  may  be  fixed  by 
passing  them  five  times  through  the  flame,  but  the  method  by  burning 
aVohol  recommended  for  fixing  blood-films  gives  more  satisfactory 
bacterial  fixation.  For  routine  work  the  stain  recommended  is  a 
dilute  carbol  fuchsin.  Drop  about  five  to  ten  drops  of  water  on  the 
cover-glass,  then  add  one  drop  of  carbol  fuchsin.  Allow  the  dilute 
stain  to  act  from  one  to  two  minutes,  then  wash  in  water,  dry  between 
small  squares  of  filter-paper  (4x4  in.),  and  mount  in  balsam  or  the 
oil  used  for  the  1/12  in.  immersion  objective.  Some  prefer  to  mount 
directly  in  water  without  preliminary  drying.  It  is  good  practice  to 
make  a  rule  to  always  keep  the  smeared  side  of  the  preparation  up — 
never  allowing  it  to  be  reversed.  By  this  simple  rule,  preparations 

27 


28  STAINING    METHODS. 

can  be  carried  through  the  most  complicated  staining  methods  without 
the  necessity  of  scratching  the  cover-glass,  etc.,  to  see  which  is  the  film 
side.  In  grasping  a  cover-glass  with  a  Cornet  or  Stewart  forceps,  be 
sure  that  the  tips  are  well  by  the  margins  of  the  glass,  otherwise  the 
stain  will  drain  off.  In  staining  with  slides,  the  grease  pencil  and  the 
glass  tubing,  as  recommended  under  Blood  Smears,  will  be  found 
useful.  The  dilute  carbol  fuchsin  and  Loffler's  methylene  blue  are 
probably  the  best  routine  stains. 

Loffler's  Alkaline  Methylene  Blue. — Saturated  alcoholic  solution 
of  methylene  blue,  30  c.c.;  one  to  ten  thousand  caustic  potash  solution, 
100  c.c.  (Two  drops  of  a  10%  solution  KOH  in  100  c.c.  of  water 
makes  a  i :  10,000  solution.) 

Carbol  Fuchsin  (Ziehl-Neelsen). — Saturated  alcoholic  solution 
basic  fuchsin,  10  c.c.;  5%  aqueous  solution  carbolic  acid,  100  c.c. 

Gram's  Method. — The  most  important  staining  method  in  bac- 
teriological techni:  and  the  one  so  rarely  giving  satisfactory  results  to 
the  inexperienced  is  Gram's  stain.  In  using  this  method,  the  lollowing 
points  must  be  kept  in  mind: 

1.  Laboratory  cultures   (subcultures)     which   have  been  carried 
over  for  years  frequently  lose  their  Gram  characteristics. 

2.  Cultures  which  are  several  days  old  or  dead  or  degenerated  do 
not  stain  characteristically. 

3.  The  aniline  gentian  violet  deteriorates  when  exposed  to  light 
in  two  or  three  days — it  should  be  kept  in  the  dark.     It  should  have  a 
rich,  creamy,  violet  appearance. 

4.  The  iodine  solution  deteriorates  and  becomes  light  in  color. 
It  should  be  of  a  rich  port-wine  color. 

5.  The  decolorizing  with  95%  alcohol  -should  stop  as  soon  as  no 
more  violet  stain  streams  out.     This  is  best  observed  over  a  white 
background,  washing  at  intervals.     Do  not  confuse  stain  on  forceps 
for  that  on  preparation. 

6.  The  preparation   should  be   thin  and  evenly  spread.     Some 
prefer  carbol  gentian  violet   to   aniline   gentian   violet.     (Saturated 
alcoholic  solution  of  gentian  violet,  one  part;  5%  aqueous  solution  of 
carbolic  acid,  ten  parts.)     This  tends  to  overstain.     The  following 
stock  solutions  of  Weigert  are  recommended: 


GRAMS    METHOD.  29 

No.  i.  No.  2. 

Gentian  violet,  2  grams.  Gentian  violet,         2  grams. 

Aniline  oil,  9  c.c.  Distilled  water,  100  c.c. 

Alcohol  (95%),  33  c.c. 

These  stock  solutions  keep  indefinitely.  Mix  i  c.c.  of  No.  i 
with  9  c.c.  of  No.  2.  Filter.  This  keeps  about  two  weeks  and  is 
the  solution  to  pour  on  the  preparation.  It  may  be  kept  on  from  two 
to  five  minutes.  Some  hasten  the  staining  by  steaming  as  for  tubercle 
bacilli.  Next  wash  the  preparation  with  water  and  flood  the  cover- 
glass  with  Gram's  iodine  solution.  Some  bacteriologists  simply  pour 
off  excess  of  aniline  gentian  violet  and  immediately  drop  on  the  iodine 
solution.  It  is  well  to  repeat  the  application  of  the  iodine  solution  a 
second  time.  The  iodine  solution  is  left  on  one  minute  or  until  the 
preparation  has  a  coffee-grounds  brown  color. 

Gram's  Iodine  Solution. 
Iodine,  i  gram. 

Potassium  iodide,     2  grams. 
Distilled  water,     300  c.c. 

After  washing  off  the  excess  of  iodine  solution  at  the  tap,  drop  on 
95%  alcohol  and  decolorize  until  no  more  violet  color  streams  out. 
Now  wash  again  and  counterstain  either  with  the  dilute  carbol  fuchsin 
or  with  a  saturated  aqueous  solution  of  Bismarck  brown. 

The  Gram  positive  bacteria  are  stained  a  deep  violet-black. 

Stained  by  Gram's  method.  Not  stained  by  Gram's  method. 

S.  pyogenes  aureus.  Meningococcus. 

S.  pyogenes  albus.  M.  catarrhalis. 

S.  pyogenes.  M.  melitensis. 

M.  tetragenus.  B.  typhosus. 

Pneumococcus.  B.  coli  communis. 

Anthrax  bacillus.  B.  dysenteriae  (Shiga). 

Tubercle  bacillus.  Sp.  cholerae  asiaticae. 

Lepra  bacillus.  B.  pyocyaneus. 

Tetanus  bacillus.  B.  mallei. 

Diphtheria  bacillus.  B.  pneumoniae  (Friedlander) . 


30  STAINING    METHODS. 

Stained  by  Gram's  method.  Not  stained  by  Gram's  method. 

B.  aerogenes  capsulatus.  B.  proteus. 

Odium  albicans.  B.  of  influenza. 

Mycelium  of  actinomyces.  B.  of  bubonic  plague. 

B.  of  chancroid. 

B.  of  Koch- Weeks. 

Gonococcus. 

Method  for  Staining  Acid-fast  Bacilli. — i.  Carbol  fuchsin' 
with  gentle  steaming  for  one  to  two  minutes  or  in  the  cold  three  to 
five  minutes. 

2.  Wash  in  water. 

3.  Decolorize  in  95%  alcohol  containing  3%  of  hydrochloric  acid 
(acid  alcohol),  until  only  a  suggestion  of  pink  remains — almost  white. 

4.  Wash  in  water. 

5.  Counterstain  in  saturated  aqueous  solution  of  methylene  blue 
or  with  LofBer's  methylene  blue. 

6.  Wash,  dry  and  mount. 

A  very  beautiful  stain  for  bacteria  in  pus,  etc.,  is  Pappenheim's 
solution. 

Sat.  aqueous  sol.  methyl  green    50  c.c. 
Sat.  aqueous  sol.  pyronin.  15  c.c. 

The  bacteria  are  stained  red;  cell  nuclei  blue  to  purple. 

Smith's  formol  fuchsin. 

Saturated  alcoholic  solution  basic  fuchsin,    10  c.c. 
Methyl  alcohol,  10  c.c. 

Formalin,  *  10  c.c. 

Distilled  water  to  make  100  c.c. 

This  gives  a  very  sharp  differentiation  of  bacteria  and  nuclear 
structures.  It  has  a  purplish  tinge.  Fixation  by  heat  gives  the  best 
staining.  Allow  the  stain  to  act  for  two  to  ten  minutes.  It  should 
not  be  used  until  after  standing  twenty-four  hours,  and  after  standing 
about  two  weeks  it  appears  to  lose  its  sharp  staining  power. 


CAPSULE    STAINING.  31 

Neisser's  stain  for  diphtheria  bacilli. 

Solution  No.  i.  Solution  No.  2. 

Methylene  blue,              o.i  gram.  Bismarck  brown,                 .2 

Alcohol,                              2  c.c.  Water  (boiling),               100  c.c. 

Glacial  acetic  acid,            5  c.c.  Dissolve  the  stain  in  the  boiling 

Distilled  water,                95  c.c.  water  and  filter. 

Dissolve  the  methylene  blue 
in  the  alcohol  and  add  it  to  the 
acetic  acid  water  mixture.  Filter. 

To  stain:  Fix  the  preparation.  Pour  on  the  dilute  acetic  acid 
methylene  blue  solution  and  allow  to  act  from  thirty  to  sixty  seconds. 
Wash.  Then  pour  on  the  Bismarck  brown  solution,  and  after  thirty 
seconds  wash  off  with  water.  Dry  and  mount.  The  bodies  of  the 
bacilli  are  brown  with  dark  blue  dots  at  either  end. 

Neisser  recommends  only  five  seconds  as  the  time  of  application  of 
each  solution.  He  also  recommends  that  the  culture  be  only  nine  to 
eighteen  hours  old  and  that  the  temperature  of  the  incubator  do  not 
exceed  36°  C.  Incubation  at  37°  C.  gives  satisfactory  results. 

Formulae  for  the  Romanowrsky  stains  and  for  carbol  thionin  are 
given  in  the  section  on  blood. 

Capsule  Staining. — The  best  method  for  studying  bacteria,  as  to 
presence  of  capsules,  is  in  the  hanging  drop,  with  the  greater  part  of  the 
light  shut  off  by  the  diaphragm. 

In  material  where  capsules  are  wrell  developed,  as  in  pneumonic 
sputum,  the  Gram  method  of  staining  brings  out  the  capsule  perfectly. 
This  is  of  diagnostic  value,  as  the  more  or  less  nonpathogenic  pneu- 
mococci  common  about  the  mouth  do  not  seem  to  show  a  capsule 
when  stained  in  this  way. 

The  most  beautiful  method  of  staining  capsules  is  the  latest  one 
proposed  by  Muir. 

1.  Prepare  thin  film,-  dry  and  stain  in  carbol  fuchsin  one-half 
minute;  the  preparation  being  gently  heated  (steamed). 

2.  Wash  slightly  in  95%  alcohol,  then  wash  well  afterward 
in  water. 

3.  Flood  preparation  in  mordant  for  five  to  ten  seconds. 


32  STAINING    METHODS. 

Mordant. — Sat.  aqueous  sol.  mercuric  chloride,  2  parts 
Tannic  acid  (20%  aqueous  sol.),  2  parts 
Sat.  aqueous  sol.  potash  alum,  5  parts 

4.  Wash  in  water  thoroughly. 

5.  Treat  with  95%  alcohol  for  one  minute.     (The  preparation 
should  have  a  pale  red  color.) 

6.  Wash  well  in  water. 

7.  Counterstain  with  methylene  blue  one-half  minute. 

8.  Dehydrate  in  alcohol.     Clear  in  xylol  and  mount.     (May 
simply  dry  specimen  with  filter-paper.) 

Flagella  Staining. — Inoculate  a  tube  of  sterile  water  (gently)  in 
upper  part,  with  just  enough  of  an  i8-to  24-hour-old  agar  culture,  to 
produce  faint  turbidity.  Incubate  for  two  hours  at  37°  C.  From  the 
upper  part  of  culture  take  a  loopful  and  deposit  it  on  a  cover-glass. 
Dry  in  thermostat  for  one  to  five  hours  or  over  night.  Use  perfectly 
clean  cover-glasses.  To  stain  by. 

Muir's  Modified  Pitfield  Method. — i.  Flood  specimen  with 
mordant.  Steam  gently  one  minute. 

Mordant. — Tannic  acid    (10%  aqueous  solution),  10  c.c. 

Sat.  aq.  sol.  mercuric  chloride,  5  c.c. 

Sat.  aq.  sol.  alum,  5  c.c. 

Carbol  fuchsin  5  c.c. 

Allow  precipitate  to  settle  or  centrifuge.     Keeps  only  one  week. 

2.  Wash  well  in  water  for  two  minutes. 

3.  Dry  carefully — preferably  in  incubator. 

4.  Pour  on  stain.     Steam  gently  one  minute. 
Stain. — Sat.  aq.  sol.  alum,  10  c.c. 

Sat.  ale.  sol.  gentian  violet,  2  c.c. 

(May    use    carbol    fuchsin    instead    of    gentian    violet.) 
Stain  only  keeps  two  days. 

5.  Wash  well  in  water.     Dry  and  mount. 

Spore  Staining.— The  most  satisfactory  spore  staining  method  is 
really  the  negative  staining  of  the  spore  obtained  "when  a  bacterial 
preparation  is  stained  by  dilute  carbol  fuchsin  or  LofHer's  methylene 


SPORE   STAINING.  33 

blue.     The  spore  appears  as  a  highly  refractile  piece  of  glass  in  a 
colored  frame. 

The  acid-fast  method,  as  for  tubercle  bacilli,  gives  good  results. 
The  decolorizing,  however,  must  be  lightly  done,  otherwise  the  spore 
will  lose  its  red  stain. 


CHAPTER  IV. 

STUDY   AND    IDENTIFICATION   OF   BACTERIA— GENERAL 
CONSIDERATIONS. 

IN  order  to  study  bacteria  it  is  necessary  to  isolate  them  in  pure 
culture.  This  may  be  accomplished  by  taking  one  or  more  loopfuls  of 
the  material  and  mixing  it  in  a  tube  of  melted  agar  or  gelatin.  From 
this  first  tube  one  or  more  loopfuls  are  transferred  to  a  second  tube  of 
melted  agar  or  gelatin,  and  from  this  a  third  transfer  is  made,  thereby 
giving  us  tubes  in  which  the  distribution  of  the  bacteria  is  one  or  more 
hundred  times  less  in  the  second  than  in  the  first  tube,  and  equally 
more  dilute  in  the  third  than  in  the  second.  When  we  pour  the  con- 
tents of  the  tubes  into  Petri  dishes  we  would  have  the  bacterial  colonies 
on  the  first  plate  so  thick  that  it  would  be  impossible  to  pick  up  a 
single  colony  with  a  platinum  needle  without  touching  a  different  one. 
On  the  second  plate  the  distribution  might  be  such  that  we  should 
have  discrete,  well  separated  colonies,  material  from  which  could  be 
taken  up  on  the  point  of  the  needle  or  loop  without  touching  any  other 
colony.  If  the  second  plate  did  not  meet  these  requirements,  the 
third  would. 

In  clinical  bacteriology  we  work  almost  entirely  with  organisms 
preferring  blood-heat  temperature,  hence  it  is  necessary  to  use  agar 
or  blood-serum  as  standard  media  for  the  obtaining  of  isolated  colonies. 
Gelatin  is  of  little  value  for  this  purpose  in  medical  work.  In  using 
agar  it  will  be  remembered  that  it  solidifies  at  a  temperature  slightly 
below  40°  C.  and  does  not  melt  again  until  it  is  subjected  to  a  tempera- 
ture practically  that  of  boiling.  Again,  if  the  temperature  of  the 
media  exc;eeds_  44°  C.  it  may  affect  injuriously  the  organisms  we  wish 
to  study.-  Consequently  it  requires  careful  attention  and  quick  work 
to  inoculate  the  tubes,  mix,  transfer  and  pour  into  plates  within  the 
limits  of  a  temperature  which  injures  the  organisms,  and  one  which 
brings  about  the  solidification  of  the  agar. 

34 


STREAKED    PLATES. 


35 


Again,  we  not  only  have  colonies  developing  from  organisms  which 
have  been  fixed  at  the  surface  as  the  agar  solidified  in  the  plate,  but 
more  numerous  ones  developing  from  bacteria  caught  in  the  depths 


FIG.  8. — Petri  Agar  Plate.  Made  by  spreading  scrapings  from  the  mouth  over 
sterilized  nutrient  agar;  after  48  hours  in  the  thermostat  the  light  "colonies" 
develop.  Streaked  plate.  (Delafield  and  Prudden.) 

21363 

of  the  media.  Therefore  we  have  superficial  and  deep  colonies. 
Except  to  the  person  of  great  experience,  all  deep  colonies  look  alike 
and  there  is  at  times  great  difficulty  in  deciding  whether  a  colony  is 
deep  or  superficial.  It  is  in  the  matter  of  trying  to  obtain  information 


36  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

from  the  differences  in  deep  colonies  that  the  greatest  difficulties  in  the 
study  of  bacteriology  arise.  By  using  the  method  of  simply  stroking 
plates  along  five  or  six  parallel  lines  from  one  side  of  the  plate  to  the 
other  with  a  glass  rod,  loop  or  swab,  we  obtain  colonies  which  are  well 
separated  and  which  are  entirely  superficial. 

The  material  as  pus,  feces,  throat  membrane,  etc.,  should  be  evenly 
distributed  in  a  tube  of  sterile  water  or  bouillon;  the  swab  which  was 
originally  used  for  obtaining  the  material  being  then  pressed  against 
the  sides  of  the  test-tube  to  express  excess  of  fluid  and  then  stroked 
gently  over  successive  lines  on  one  plate.  Or,  if  the  organisms  be 
very  abundant,  over  a  second  plate  without  recharging  it  from  the 
inoculated  tube. 

To  obtain  isolated  colonies  on  blood-serum  or  blood-streaked  agar, 
which  can  be  touched  and  by  transfer  obtained  in  pure  culture,  we 
simply  smear  the  material  on  a  slant  of  either  medium.  Then,  without 
sterilizing  the  loop,  we  smear  it  thoroughly  over  a  second  slant,  and  so  on 
to  a  third,  or  possibly  a  fourth  or  fifth. 

At  present  the  classification  of  the  bacteria  is  very  unsatisfactory 
from  a  scientific  stand-point.  The  nomenclature  abounds  in  instances 
where  three  or  four  terms  are  used  in  naming  a  single  bacterium,  in- 
stead of  the  single  generic  name  and  single  specific  one  as  is  used  in 
zoological  nomenclature.  This  matter  of  nomenclature  is  a  subor- 
dinate factor  in  the  confusion  when  we  begin  to  investigate  and  find 
that  different  names  have  been  applied  to  apparently  the  same  organ- 
ism. The  slightest  variation  in  morphological,  locomotor  or  biological 
characteristics  seems  to  be  considered  sufficient  by  many  observers  to 
justify  the  description  of  a  new  species,  and,  of  course,  the  giving  of 
a  new  name.  Many  of  these  names  which  are  now  retained  were 
applied  prior  to  the  epoch-making  introduction  of  gelatin  media  by 
-Koch  (1881)  and  consequently  at  a  time  when  the  isolation  of  organisms 
in  pure  culture  was  a  matter  of  extreme  difficulty  and  uncertainty. 
One  of  the  first  facts  noted  by  the  student  in  taking  up  bacteriology  is 
the  difficulty  in  determining  motility;  this  property  should  always  be 
tested  on  young  cultures  in  bouillon.  In  Brownian  movement  there 
is  a  sort  of  scintillating  movement,  but  the  bacterium  does  not  move 
from  that  part  of  the  field.  In  current  movement  all  the  bacteria 


CULTURAL   CHARACTERISTICS. 


37 


swarm  in  the  same  direction,  going  very  fast  at  times,  and  then  more 
slowly.  If  in  great  doubt,  the  mounting  of  the  organisms  in  a  2% 
solution  of  carbolic  acid  will  stop  movement  if  it  be  true  functional 
motility,  while  Brownian  and  current  movement  are  not  interfered 


Chflncl'Vo 


FIG.  9. — Chart  in  use  at  the  U.  S.  Naval  Medical  School. 

with.  In  true  motility  bacteria  move  in  opposite  and  in  all  directions, 
and  move  away  from  the  place  where  first  observed  unless  degenerated 
or  dead. 

Reaction  of  media  is  of  the  greatest  importance  in  causing  variation 
in  the  functions  of  bacteria,  and  is  one  which  has  until  recently  been 


38  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

almost  entirely  neglected.  In  describing  an  organism  at  the  present 
time  it  is  always  necessary  to  note  the  reaction  of  the  media,  the  tem- 
perature at  which  cultivation  took  place  and  the  age  of  the  culture 
when  examined. 

In  the  following  keys  the  term  bacterium  has  been  used  as  a  general 
designation  for  all  schizomycetes.  Migula  calls  motile  rod-shaped 
organisms  bacilli,  and  nonmotile  ones  bacteria.  Lehmann  and 
Neumann  call  spore-bearing  organisms  bacilli,  and  nonspore-bearing 
ones  bacteria. 

The  B.  typhosus  is  very  motile  and  does  not  possess  spores.  Ac- 
cording to  Migula,  it  would  be  the  Bacillus  typhosus;  according  to 
Lehmann  and  Neumann,  the  Bacterium  typhosum.  The  B.  anthracis 
has  spores  and  is  nonmotile.  Hence  it  would  be  Bacterium  anthra- 
cis, according  to  Migula,  and  the  Bacillus  anthracis,  according  to 
Lehmann  and  Neumann. 

In  the  use  of  the  keys  at  the  head  of  each  group  of  organisms  it  will 
be  observed  that  the  primary  separation  is  on  the  basis  of  morphology 
— the  cocci  in  one  group,  the  bacilli  in  three  subgroups:  one  for  those 
rod-shaped  organisms  showing  branching  and  curving  forms,  one  for 
the  spore  bearers  and  one  for  the  simple  rods.  The  spirilla  are 
grouped  by  themselves. 

An  important  method  of  differentiation  is  the  reaction  to  Gram's 
stain.  It  should  be  remembered  that  organisms  carried  along  on 
artificial  media  often  lose  their  Gram  staining  characteristics;  hence  it 
is  desirable  to  determine  this  staining  reaction  in  cultures  freshly 
isolated.  Be  sure  that  the  stains,  especially  the  aniline  gentian  violet 
and  the  iodine  solution,  have  not  deteriorated.  There  is  no  more 
important  stain  than  this,  and  none  which  requires  greater  experience. 
The  chief  causes  of  conflicting  results  are  (i)  working  with  old  cultures 
and  (2)  not  having  satisfactory  staining  solutions. 

Motility,  as  stated  above,  is  at  times  difficult  to  determine.  For  this 
purpose  young  eighteen-hour-old  bouillon  cultures  are  preferable,  and 
the  preparation  should  be  made  by  applying  a  vaselin  ring  to  the 
slide,  then  putting  a  drop  of  the  bouillon  culture  in  the  center  of  the 
ring  (or  a  drop  of  water  inoculated  from  an  agar  slant  growth),  then 
putting  on  a  cover-glass.  By  this  method  current  movement  is  done 


CULTURAL   CHARACTERISTICS.  39 

away  with  and  the  preparation  keeps  for  hours.     This  is  a  convenient 
method  for  agglutination  tests. 

Liquefaction  of  gelatin  is  a  very  important  means  of  differentia- 
ting. When  a  room-temperature  incubator  is  not  at  hand  (20°  to  22°  C.), 
it  is  better  to  put  the  inoculated  gelatin-tube  in  the  body-temperature 


FIG.  10. — Series  of  stab  cultures  in  gelatine,  showing  modes  of  growth  of 
different  species  of  bacteria.     (Abbott.) 

incubator,  and  from  day  to  day  test  the  power  of  solidifying  with  ice- 
water.  If  the  organism  digests  the  gelatin  (a  liquefier),  the  medium 
will  remain  fluid  when  placed  in  ice-water — if  the  organism  is  a  non- 
liquefier,  the  medium  in  the  tube  becomes  solid.  Of  course  we  lose  the 
information  to  be  obtained  from  the  shape  of  the  area  of  liquefaction. 


40  STUDY   AND    IDENTIFICATION    OF    BACTERIA. 

For  routine  work  the  only  sugar  media  used  are  the  glucose  and  the 
lactose  bouillon.  These  are  of  the  utmost  importance  in  differentia- 
ting organisms  of  the  typhoid  and  colon  group.  Following  Ford,  these 
intestinal  bacteria  have  primarily  been  separated  by  their  action  on 
litmus  milk — whether  turning  it  pink  or  only  slightly  changing  or 
not  changing  at  all  the  original  color. 


CHAPTER  V. 

STUDY  AND  IDENTIFICATION  OF  BACTERIA— COCCI. 
KEY  AND  NOTES. 

Streptococcus  Forms. — Cells  divide  to  form  chains. 

A.  Gelatin  not  liquefied. 

1.  Tendency  to  form  long  chains. 

a.  Streptococcus  pyogenes.     (Cocci  .7  to  i  /*.) 

b.  Streptococcus  anginosus. 

2.  Tendency  to  form  short  chains. 

a.  Streptococcus  salivarius. 

b.  Streptococcus  fecalis. 

B.  Gelatin  liquefied. 

a    Streptococcus  coli  gracilis.     (Cocci  quite  small — .2  to  .4/1.     In  feces.) 
Sarcina  Forms. — Cells  divide  in  three  dimensions  of  space.     (Packets.) 

A.  No  pigment  production  on  agar. 

a.  Sarcina  alba.     (Colonies  finely  granular.) 

b.  Sarcina  pulmonum. 

B.  Yellowish  pigment. 

a.  Sarcina  lutea.     (Colonies  coarsely  granular.) 

b.  Sarcina  flava.     (Colonies  finely  granular.) 

C.  Rose-red  pigment. 

a.  Sarcina  rosea. 
Micrococcus  Forms. — Cells  divide  irregularly  in  various  directions. 

I.  Gram  positive  cocci. 

A.  Cocci — round. 

1.  Divide  in  two  planes  at  right  angles.     Merismopedia. 

a.  M.  tetragenus.     Moist    viscid    colonies.     No    liquefaction    of 
gelatin.     Capsule. 

2.  Divide  irregularly. 

a.  Gelatin  not  liquefied.     M.  cereus  albus. 

h    Pelatin    linuefied  I  M.  (Staphylococcus}  pyogenes  albus. 

b.  O  lique  ed.  J  M   (StaphyoIocccus)   pvogenes  aureus. 

c.  Gelatin  very  slightly  liquefied. 

S.  epidermidis  albus.     (Stitch  coccus.) 

B.  Cocci — biscuit-shape. 

Diplococcus  crassus.     (May  be  mistaken  for  meningococcus.) 

C.  Cocci — lance  shape. 

Diplococcus  pneumoniae.     Fraenkel's  pneumococcus.     Capsule. 

II.  Gram  negative  cocci. 

A.  Grow  only  about  incubator  temperature. 

1.  Grow  only  on  blood  or  serum  media.     Gonococcus. 

2.  Grow  on  blood  serum  media,  or  glycerine  agar. 

a.  Diplococcus   intracellularis   meningitidis.     (Produces   acid    in 
glucose.) 

3.  Grows  on  ordinary  media.     Micrococcus  melitensis. 

B.  Will  grow  at  room  temperature  as  well  as  at  37°  C. 

a.  Micrococcus  catarrhalis.     (Produces  alkalinity  in  glucose  media.) 

41 


42  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

STREPTOCOCCUS  FORMS. 

Those  cocci  tending  to  arrange  themselves  in  chains  are  usually 
described  as  streptococci.  When  we  consider  that  certain  bacilli  at 
times  assume  an  arrangement  which  we  term  strepto-bacilli,  yet  have 
no  relationship,  it  would  suggest  that  the  matter  of  chain  morphology 
is  simply  a  characteristic  common  to  many  entirely  different  cocci. 

The  essential  point  to  bear  in  mind  is  that  the  finding  of  a  strepto- 
coccus does  not  necessarily  explain  an  infection,  because  normally 
streptococci  are  among  the  organisms  most  frequently  and  abundantly 
found  in  plates  made  from  normal  buccal  and  nasal  secretions.  It  is 


FIG.  ii. — Streptococcus  pyogenes.     (Kolle  and  Wassermann.) 

well  to  be  very  conservative  when  reporting  streptococci  as  the  etiologi- 
cal  factor  from  lesions  of  the  throat  or  nose. 

Probably  the  most  practical  point  in  the  differentiation  of  strepto- 
cocci, next  to  that  of  pathogenicity,  is  the  occurrence  of  long  or  short 
chains,  the  virulent  ones  tending  to  appear  in  chains  of  from  ten  to 
twenty  cocci,  while  the  normal  inhabitants  of  the  nose,  mouth  and 
feces  generally  tend  to  be  in  shorter  chains. 

As  regards  virulence,  this  is  exceedingly  variable — it  is  soon  lost,  but 
may  be  restored  either  by  inoculating  streptococci  along  with  various 
other  organisms  or  by  passage  through  successive  rabbits.  The  rabbit 
is  the  most  susceptible  animal  and  should  be  inoculated  in  one  of  the 


STREPTOCOCCI.  43 

prominent  ear  veins.  If  the  needle  of  the  syringe  is  not  inserted  in  the 
vein  it  will  be  difficult  to  force  in  the  material  and  a  swelling  will  im- 
mediately show  itself. 

Besides  the  morphological  and  pathogenic  variations,  Schottmuller 
has  noted  differences  where  these  organisms  are  grown  on  one  part  of 
blood  and  three  parts  of  agar.  On  this  medium  Strep,  erysipelatis  has 
a  hemolytic  action,  the  laking  of  the  red  cells  bringing  about  a  more  or 
less  clear  ring  surrounding  the  colony.  The  short- chain  streptococci 
do  not  have  a  hemolytic  halo. 

Some  of  the  English  authorities  have  introduced  biochemical 
methods  of  differentiating:  the  Strep,  pyogenes  coagulating  milk, 
reducing  neutral  red,  and  producing  acid  in  lactose,  saccharose, 
mannite  or  inulin  media.  When  we  consider  the  biochemical  varia- 
tions which  a  single  organism,  as  the  colon  bacillus,  may  exhibit,  the 
value  of  such  methods  of  differentiating  may  well  be  questioned.  The 
question  of  the  symbiotic  relationship,  which,  when  established  between 
two  or  more  bacteria,  may  cause  harmless  organisms  to  take  on  viru- 
lence, would  appear  to  be  a  more  important  consideration. 

Almost  without  exception,  streptococci  are  .Gram  positive.  Their 
colonies  are  quite  small,  but  distinct  and  discrete.  In  appearance  the 
colonies  of  streptococci  and  pneumococci  are  practically  identical.  In  a 
blood- serum  throat  culture  pneumococcus  and  streptococcus  colonies 
are  the  smallest,  diphtheria  ones  are  quite  small  and  discrete,  but  slightly 
flatter.  (Always  examine  the  water  of  condensation  for  streptococci.) 
The  sarcina  and  staphylococcus  colonies  are  much  larger. 

Streptococci  are  commonly  the  cause  of  diffuse  phlegmonous  in- 
flammation, while  the  staphylococci  cause  circumscribed  lesions. 
Streptococci  cause  necrosis  and  do  not  characteristically  produce  pus. 
The  importance  of  the  streptococcus  as  a  secondary  infection  in  diph- 
theria, tuberculosis,  small-pox  and  even  in  typhoid  fever  must  always 
be  kept  in  mind.  It  is  this  infection  which  does  not  respond  to  diph- 
theria antitoxin,  and  not  the  diphtheria  one. 

SARCINA  FORMS. 

These  are  best  observed  in  hanging-drop  preparations,  when  they 
can  be  seen  as  little  cubes,  like  a  parcel  tied  with  a  string,  and  by  noting 


44  STUDY   AND    IDENTIFICATION    OF    BACTERIA. 

them  when  turning  over,  it  will  be  seen  that  they  are  different  from  the 
tetrads  which  only  divide  in  two  directions  of  space.  At  times  the 
packet  formation  is  not  perfect  and  it  will  be  difficult  to  distinguish 
such  as  sarcinae.  All  sarcinae  stain  by  Gram.  If  the  staining  of 
sarcinse  be  too  deep  it  may  obscure  the  lines  of  cleavage.  Sarcinaae  are 
nonmotile. 

Various  sarcinae  have  been  isolated  from  the  stomach,  especially 
when  there  is  stagnation  of  stomach  contents.  Sarcinae  have  also  been 
found  in  the  intestines.  In  plates  the  S.  lutea  is  frequently  a  contami- 
nating organism,  being  rather  constantly  present  in  the  air.  The 
demonstration  of  sarcina  morphology  should  always  be  made  from 
liquid  media,  as  bouillon.  Urine  makes  an  excellent  medium. 

MICROCOCCUS  FORMS. 

This  grouping  includes  all  cocci  which  do  not  show  chain  or  packet 
formation.  It  will  be  found  convenient  to  divide  them  into  two  classes 
according  to  their  staining  by  Gram.  The  M.  tetragenus,  S.  pyogenes 
aureus  and  the  pneumococcus  stain  by  Gram,  while  the  gonococcus 
the  meningococcus,  the  M.  catarrhalis  and  the  M.  melitensis  are 
Gram  negative. 

M.  Tetragenus. — This  organism  is  frequently  found  associated 
with  other  organisms  in  sputum,  especially  with  tubercle  and  influenza 
bacilli.  The  colonies  are  white,  slightly  smaller  than  staphylococci  and 
are  quite  viscid. 

It  was  formerly  considered  unimportant  in  disease,  but  the  idea  now 
prevails  that  it  is  responsible  for  many  abscesses  about  the  mouth, 
especially  in  connection  with  the  teeth.  Injected  subcutaneously  into 
mice,  it  produces  a  septicaemia  and  death  in  three  or  four  days.  The 
blood  shows  great  numbers  of  encapsulated  tetrads.  It  has  been  re- 
ported twice  as  a  cause  of  septicaemia  in  man. 

Staphylococci. — To  cocci  dividing  irregularly  and  usually  forming 
masses  which  are  likened  to  clusters  of  grapes  the  term  staphylococcus 
is  applied.  While  there  have  been  experiments  which  show  that  by 
selecting  pale  portions  of  a  yellow  colony,  eventually  a  white  colony 
could  be  produced,  yet,  as  a  practical  consideration,  it  is  convenient 
to  consider  at  least  two  types  of  staphylococci:  the  staphylococcus 


STAPHYLOCOCCI. 


45 


pyogenes  aureus  and  the  staphylococcus  pyogenes  albus.  In  culturing 
from  the  pus  of  an  abscess  or  furuncle  we  generally  obtain  a  golden 
coccus,  while  in  material  from  the  nose  or  mouth,  the  staphylococcus 
colonies  are  almost  invariably  white.  As  regards  the  common  skin 
coccvs,  this  will  be  found  to  produce  a  white  colony.  A  coccus  which 
very  slowly  liquefies  gelatin  and  has  been  sup- 
posed to  cause  stitch  abscesses  is  the  S.  epi- 
dermidis  albus. 

While  it  is  customary  to  look  for  a  golden 
colony  in  the  case  of  organisms  showing 
virulence,  yet  at  times  a  cream -white  colony 
may  develop  from  cocci  of  great  virulence. 

The  S.  pyogenes  citreus  is  considered  as  of 
very  feeble  pathogenic  power.  Certain  cocci 
whose  colonies  have  presented  a  waxy  appear- 
ance have  been  designated  as  S.  cereus  albus 
and  S.  cereus  flavus,  respectively.  They  are  of 
very  little  practical  importance.  The  staphylo- 
coccus pyogenes  aureus  grows  readily  at  room 
temperature,  but  better  at  37°  C.  It  coagulates 
milk  and  renders  bouillon  un'formly  turbid. 
It  grows  on  all  media,  as  blood-serum,  agar, 
potato,  etc.  It  has  been  proposed  to  distin- 
guish it  from  skin  staphylococci  by  its  power 
of  producing  acid  in  mannite.  Ordinarily 
the  individual  cocci  are  about  la  in  diameter, 
but  they  vary  greatly  in  size  according  to  the 
age  of  the  culture  and  other  conditions.  The 
aureus,  as  it  is  frequently  called,  is  not  only 
often  found  in  circumscribed  processes,  but  it 
is  a  frequent  cause  of  septicaemia,  osteomyelitis,  endocarditis,  etc.  It  is 
the  organism  most  frequently  concerned  in  terminal  infections.  The 
lowered  resistance  of  the  patient  permits  of  their  passage  through  bar- 
riers ordinarily  resistent.  Not  only  should  this  be  kept  in  mind  when 
such  organisms  are  isolated  at  an  autopsy,  but  as  well  the  fact  that  their 
entrance  may  have  been  agonal  or  subsequent  to  death. 


FIG.  12. — Gelatine  cul- 
ture Staphylococcus 
aureus  one  week  old. 
(Williams.) 


46  STUDY   AND    IDENTIFICATION    OF    BACTERIA. 

The  Pneumococcus  of  Fraenkel.— This  is  by  far  the  most  com- 
mon cause  of  pneumonia,  whether  it  be  of  the  croupous,  catarrhal  or 
septic  type.  It  is  also  frequently  found  in  meningitis,  empyema, 
endocarditis  and  otitis  media.  It  should  not  be  confused  with  the 
pneumobacillus  of  Friedlander,  which,  although  possessing  a  capsule 
like  the  pneumococcus,  differs  from  it  by  being  Gram  negative,  being  a 
bacillus  and  having  large  viscid  colonies.  The  pneumococcus  is  the 
cause  of  more  than  80%  of  the  cases  of  pneumonia.  It  does  not  grow 
below  20°  C.  and  is  best  cultivated  on  blood-serum,  or  blood-streaked 


. 

«*%»  , 

.;  *  - 


FIG.  13.  —  Pneumococcus,  showing  capsule,  from  pleuritic  fluid  of  infected 
rabbit,  stained  by  second  method  of  Hiss.     (Williams.) 

agar.  On  plain  agar  it  grows  as  a  very  small  dew-drop-like  colony, 
which  is  slightly  grayish  by  reflected  light.  It  is  smaller  and  more 
transparent  than  a  streptococcus  colony.  In  sputum  or  other  patholog- 
ical material  it  is  best  recognized  by  the  presence  of  a  capsule  inclosed 
in  which  are  two  lance-shaped  cocci  with  their  bases  apposed.  In  arti- 
ficial culture  we  rarely  get  the  capsule.  It  also  sometimes  grows  in 
short  chains  like  a  streptococcus.  The  best  medium  for  differentiating 
is  the  serum  of  a  young  rabbit,  in  this  it  grows  as  a  diplococcus,  while 
streptococci  show  chains.  The  best  method  of  isolating  it  in  pure 


GRAM   NEGATIVE   COCCI.  47 

culture  is  to  inject  the  sputum  into  the  marginal  ear  vein  of  a  rabbit  or 
subcutaneously  into  a  mouse.  Death  results  from  septicaemia  in 
about  two  days  and  the  blood  teems  with  pneumococci.  Usually  the 
pneumococcus  quickly  loses  its  virulence,  and  also  dies  out  in  a  few 
days  unless  transferred  to  fresh  media.  The  best  medium  for  it 
preservation  is  rabbit's  blood  agar;  this  also  maintains  the  virulence. 
On  this  medium  the  colonies  are  larger  than  on  agar  and  they  present 
a  greenish  appearance  with  a  hemolytic  zone.  It  is  a  well-known  fact 
that  the  pneumococcus  is  a  frequent  inhabitant  of  the  nasal  and  buccal 
cavities.  The  explanation  of  infection  is  either  on  the  ground  of 
lowered  resistance  of  the  patient  or  enhanced  virulence  of  the  organ- 
ism. Oscar  Richardson  has  reported  an  organism  in  cases  of  lobar 
pneumonia,  cerebrospinal  meningitis,  mastoid  disease,  etc.,  bearing 
resemblance  to  both  pneumococci  and  streptococci — the  Streptococcus 
capsulatus.  It  differs  from  the  pneumococcus  in  that  the  colonies  on 
blood-serum  are  viscid  and  like  irregular  flecks  of  mucus.  The 
characteristic  culture  is  a  glucose  agar  stab.  (Reaction  must  not 
exceed  +  .5.)  From  the  line  of  puncture  there  are  flail  like  projections 
extending  outward  from  one-fifth  to  one-fourth  of  an  inch.  The 
capsule  persists  on  culture  media.  This  organism  resembles  the 
Streptococcus  of  Bonome  of  the  French. 

Diplococcus  Crassus. — This  is  a  Gram  positive,  biscuit-shaped 
diplococcus,  which  might  be  confused  with  the  M.  catarrhalis  or  the 
meningococcus  by  ordinary  staining  methods.  In  throat  cultures  I 
have  isolated  on  several  occasions  a  Gram  positive  diplococcus  which 
is  at  times  biscuit-shaped,  at  times  irregularly  spherical.  It  possesses 
two  or  three  metachromatic  granules,  so  that  in  a  Neisser  stain  for 
diphtheria  the  appearance  of  these  granules  may  be  confusing. 

Gram  Negative  Cocci. — It  is  important  to  bear  in  mind  that  there 
are  many  cocci  of  varying  shapes,  which  in  cultures  or  in  smears  from 
the  throat,  nose  or  feces  are  Gram  negative.  These  are  not  well 
classified  or  described.  To  distinguish  the  three  important  biscuit- 
shaped  diplococci,  it  can  be  most  easily  accomplished  by  cultural 
methods,  using  hydrocele  agar  (ascites  or  blood  agar  will  answer), 
ordinary  blood-serum  and  plain  agar.  The  gonococcus  will  only 
grow  on  the  hydrocele  agar;  the  meningococcus  will  grow  on  this,  but 


48  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

likewise  grows  on  ordinary  blood-serum.     The  M.  catarrhalis  will 
grow  on  plain  agar  as  well  as  on  the  other  media. 

Gonococcus  (Neisser,  1879). — This  organism  is  characteristically 
a  diplococcus,  the  separate  cocci  being  plano-convex  with  their  plane 
surfaces  apposed.  (Biscuit  shape,  coffee-bean  shape.)  They  are 
generally  found  grouped  in  masses  of  several  pairs,  most  strikingly  in 
pus  cells  or  epithelial  cells,  but  also  found  extracellularly.  Except  in  the 
height  of  the  disease,  there  is  a  great  tendency  for  the  organisms  to  show 
involution  forms,  so  that  instead  of  biscuit-shaped  diplococci  we  have 


FIG.  14. — Gonococcus.     Film  from  urethral  pus.     (Coplin.) 

round,  irregular  and  uneven  cocci.  It  is  therefore  advisable  in  search- 
ing smears  from  chronic  gonorrhoea  to  continue  the  search  of  Gram 
stained  specimens  until  some  fairly  typical  diplococci  are  found. 
There  is  nothing  requiring  greater  discrimination  than  a  diagnosis 
from  such  a  smear.  At  the  commencement  of  a  gonorrhcea  the 
epithelial  cells  are  abundant  and  gonococci  are  found  adhering  to  them 
or  lying  free.  Later  on,  at  the  acme  of  the  discharge  (the  creamy, 
abundant  discharge),  it  is  in  the  pus  cells  we  find  them  and  they  may  be 
so  abundant  that  10  to  20%  of  the  pus  cells  may  contain  them.  In 


GONOCOCCUS.  49 

the  subacute  stage  the  epithelial  cells,  which  practically  disappear 
when  the  discharge  is  so  abundant,  begin  to  reappear,  and  in  the 
chronic  stage  the  epithelial  cells  are  the  chief  ones,  and  are  the  ones, 
in  which  we  find  an  occasional  gonococcus,  often  distorted  in  shape. 

The  best  method  of  diagnosis  in  cases  of  chronic  gonorrhoea  is  to 
have  the  patient  drink  beer  and  eat  the  stimulating  food  previously 
interdicted,  inject  a  weak  solution  of  silver  nitrate  and  massage  the 
prostate  or  seminal  vesicles.  The  smears  made  from  the  resulting 
discharge  or  centrifuged  urine  will  probably  contain  gonococci  if  they 
are  present  in  the  urethra.  In  the  female  the  favorite  sites  are  the 
urethra  and  the  cervix  uteri.  In  municipal  examinations  it  is  custom- 
ary to  make  two  smears:  one  from  the  urethral  meatus  and  a  second 
from  the  cervix.  The  vagina  is  not  a  suitable  soil  for  their  develop- 
ment. In  female  children  it  is  most  often  found  in  the  discharge  of 
the  vulvovaginitis. 

In  addition  to  the  genital  organs,  the  gonococcus  may  at  times 
invade  and  be  isolated  from  the  eye  (gonorrhceal  ophthalmia),  the 
joints,  rarely  as  a  cause  of  endocarditis  and  possibly  as  the  factor  in 
septicaemia.  Grown  upon  hydrocele  or  ascites  agar,  or  blood  streaked 
agar,  or  upon  blood  agar  from  man  or  the  rabbit,  the  colonies  appear 
as  irregular,  minute,  dew-drop  spots.  By  the  second  or  third  day  the 
involution  forms  are  abundant,  and  within  four  to  seven  days  the 
culture  will  probably  be  found  to  be  dead.  Unless  frequent  transfers 
are  made,  it  will  be  best  kept  alive  on  blood  agar.  The  organism 
grows  best  at  37°  C,  and  will  not  grow  below  25°  C.  It  will  not  grow 
on  plain  or  glycerin  agar  or  ordinary  blood-serum  unless  the  transfer 
of  considerable  pus  in  inoculating  the  slants  give  it  a  suitable  culture 
medium.  In  material  from  joints,  it  is  in  the  fibrin  flakes  that  the 
gonococci  are  most  apt  to  be  found,  if  found  at  all. 

Diplococcus  IntracellularisMeningitidis  (Weichselbaum,  1887). 
—This  is  the  organism  of  epidemic  cerebrospinal  meningitis,  and  is 
frequently  termed  the  meningococcus.  The  diplococcus  is  Gram 
negative  and  biscuit  shaped  and  is,  like  the  gonococcus,  chiefly  con- 
tained in  pus  cells.  It  is  also  found  free  in  the  cerebrospinal  fluid 
withdrawn  from  spotted  fever  cases.  There  is  a  greater  tendency  to 
variation  in  size  and  shape  than  is  the  case  with  the  gonococcus,  which 
4 


50  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

latter,  in  fresh  material,  show  a  striking  uniformity  morphologically. 
The  meningococcus  is  at  times  not  abundant — early  in  the  case,  how- 
ever, the  picture  may  be  similar  to  that  of  gonorrhoea. 

On  blood- serum  the  colonies  appear  after  24  to  48  hours  as  discrete, 
very  slightly  hazy  colonies,  about  one-eighth  of  an  inch  in  diameter. 
On  serum  agar,  as  ascites  or  hydrocele  agar,  they  grow  best.  Unless 
considerable  cerebrospinal  fluid  is  transferred  with  the  inoculating 
loop,  they  do  not  grow  on  plain  agar.  They  will  grow  at  times  on 
glycerin  agar.  It  ferments  dextrose  and  only  grows  at  blood  temper- 


FIG.  15. — Diplococcus  intracellularis  meningitidis  and  pus-cells 
(X  1000.)      (Williams.} 

ature,  thus  distinguishing  it  from  the  M.  catarrhalis.  It  is  scarcely 
pathogenic  for  laboratory  animals  when  injected  subcutaneously. 
Intradural  injections  give  results.  The  cultures  die  out  very  rapidly, 
so  that  it  is  necessary  to  make  transfers  every  one  or  two  days.  The 
meningococcus  has  been  isolated  from  the  nasal  secretions  of  patients. 
The  possibility  of  these  organisms  being  the  M.  catarrhalis  must  be 
considered. 

The  meningococcus  has  very  slight  resistance  to  sun  or  drying  so 
that  its  aerial  transmission  seems  doubtful.  It  is  supposed  to  effect 
an  entrance  by  the  nares,  thence  reaching  the  cerebral  meninges. 
Infection  is  probably  by  direct  contagion.  Several  cases  have  been 


MENINGOCOCCUS.  51 

reported  where  with  a  high  leukocytosis  the  cocci  have  been  found  in 
the  polymorphonuclears  of  blood  smears  and  in  cultures  from  the 
blood. 

By  the  use  of  alternate  injections  into  horses  of  living  diplococci, 
then  seven  days  later  of  an  autolysate  made  from  different  strains; 
seven  days  later  again  injecting  living  diplococci;  thus  alternating 
material  every  week,  an  antiserum  of  value  has  been  obtained  by 
Flexner.  The  immunization  requires  about  one  year.  In  using, 
withdraw  about  20  c.c.  of  cerebrospinal  fluid  with  a  syringe,  and  then 
inject,  through  the  same  needle,  an  equal  quantity  of  the  serum. 
The  injection  is  repeated  every  day  for  three  or  four  days. 

For  diagnosis,  make  smears  and  cultures  from  cerebrospinal  fluid. 
The  sediment  from  the  centrifuged  material  gives  better  results.  In 
tuberculosis  the  lymphocytes  preponderate ;  in  cerebrospinal  meningitis 
the  polymorphonuclears. 

It  has  been  stated  that  a  point  of  difference  between  the  phagocyto- 
sis with  the  gonococci  and  the  meningococci  is  that  the  meningococci 
invade  and  at  times  destroy  the  nucleus  of  the  polymorphonuclear, 
which  is  not  true  of  gonococci.  The  appearance  of  large  phagocytic 
endothelial  cells,  often  containing  polymorphonuclears,  in  the  centri- 
fuged cerebrospinal  fluid  is  a  favorable  prognostic  sign.  At  times 
there  does  not  appear  to  be  any  relation  between  the  number  of  pha- 
gocytic polymorphonuclears  and  the  severity  of  the  case. 

Micrococcus  Catarrhalis  (Seifert,  1890).— This  organism  has 
been  specially  studied  by  Lord.  It  resembles  the  meningococcus 
strikingly  and  can  only  be  differentiated  by  cultural  procedure.  It 
grows  on  plain  agar  and  at  room  temperature,  and  does  not  produce 
acid  in  glucose  media.  It  not  only  occurs  in  the  nasal  secretions  of 
healthy  people,  but  appears  to  be  responsible  for  certain  coryzas 
resembling  influenza.  It  also  is  responsible  for  certain  epidemics  of 
conjunctivitis.  The  colonies  are  larger,  more  opaque  and  have  a 
more  irregular  wavy  border  than  the  round  colonies  of  the  meningo- 
coccus. 

Micrococcus  Melitensis  (Bruce,  1887).— This  is  the  organism 
of  Malta  or  Mediterranean  fever,  sometimes  called  undulating  fever, 
on  account  of  successive  waves  of  pyrexia  running  over  several  months. 


52  STUDY    AND    IDENTIFICATION    OF    BACTERIA 

The  djsease  has  a  very  slight  mortality  (2%),  and  the  lesions  are  chiefly 
of  the  spleen,  which  is  large  and  diffluent.  The  organisms  can  best  be 
isolated  from  the  spleen. 

M.  melitensis  is  only  about  .3^  in  diameter.  The  characteristics 
are  its  very  small  size  and  the  dew-drop  minute  colonies  on  agar, 
which  at  incubator  temperature  only  show  themselves  about  the  third 
to  the  sixth  day.  It  is  nonmotile  and  Gram  negative.  In  bouillon 
there  is  a  slight  turbidity. 

The  organism  occurs  in  the  peripheral  circulation,  it  having  been 
cultivated  from  blood  very  successfully  by  Eyre.  He  takes  blood  at 
the  height  of  the  fever,  and  in  the  afternoon.  Formerly  it  was  custom- 
ary to  isolate  by  splenic  puncture. 

Infection  is  chiefly  by  means  of  the  milk  of  goats.  The  organisms 
are  excreted  in  the  urine  of  patients,  and  a  diagnostic  point  is  to  make 
plates  from  the  urine.  Such  urine  applied  to  abraded  surfaces  causes 
infection. 

The  serum  of  patients  shows  agglutinating  power  as  early  as  the 
fifth  day  of  the  disease,  and  this  may  persist  for  years  after  recovery. 
A  dilution  of  1 130  or  i :  50  is  recommended. 


CHAPTER  VI. 

STUDY  AND  IDENTIFICATION  OF  BACTERIA.  SPORE- 
BEARING  BACILLI.     KEY  AND  NOTES. 

A.  Grow  aerobically. 

1.  Stab  culture  in  gelatin  has  branches  growing  out  at  right  angles  to  line 
of  stab. 

a.  Has  no  membrane  on  bouillon  or  liquefied  gelatin.     Projecting 
branches  from  line  of  stab  only  at  upper  part  of  line  of  growth. 
Absolutely   nonmotile.     Ends   sharply    cut   across    or   concave. 
ANTHRAX  GROUP. 

b.  Has  thick  whitish  membrane  on  bouillon  and  surface  of  liquefied 
gelatin!     Projecting  branches  all  along  the  line  of  stab.     Sluggishly 
motile.     MYCOIDES  GROUP.     (B.  mycoides.     B.  ramosus.) 

2.  Stab  cultures  in  gelatin  do  not  show  projecting  branches. 

a.  Potato  cultures  do  not  become  wrinkled.     At  first  slightly  moist, 
later  dry  and  mealy.     SUBTILIS  GROUP.    (Hay  bacillus  group.) 
Actively  motile. 

The  yellow  subtilis  is  at  times  found  in  water.  The  colonies  on 
potato  are  of  a  cheese-yellow  color.  The  bacilli  are  very  large  and 
show  a  slugglish,  worm-like  motion. 

b.  Potato     cultures     become     wrinkled.     VULGATUS     GROUP. 
(Potato  bacillus.) 

Two  water  bacilli  belonging  to  this  group  are  the  B.  mesentericus 
fuscus  (brown  growth)  and  B.  mesentericus  ruber  (red  growth). 

NOTE. — The  following  cultural  characteristics  are  common  to  all  the  above 

spore  bearers. 

1.  Liquefaction  of  gelatin. 

2.  Milk  slowly  coagulated  with  very  little  change  in  reaction.     Later  the 
coagulum  is  digested. 

3.  No  gas  in  either  glucose  or  lactose. 

4.  No  indol. 

5.  All  are  Gram  positive. 

6.  All  digest  blood-serum. 

B.  Grow  only  anaerobically. 

1.  Rods  very  little  swollen  by  centrally  situated  spores. 

a.  Motile.     B.  cedematis     maligni.     (Gram  negative.) 

b.  Nonmotile.     B.  aerogenes  capsulatus.     (Capsule.) 

2.  Spores  tend  to  be  situated  between  center  and  end. 

a.  No  liquefaction  of  gelatin.     B.  butyricus. 

b.  Gelatin  liquefied  slowly. 

B.  botulinus.     Milk  not  coagulated. 

B.  anthracis  symptomatici. 

B.  enteritidis  sporogenes.     Milk  coagulated  with  abundant  gas. 

c.  Gelatin  liquefied  rapidly.     B.  cadaveris  sporogenes.     Very  motile. 

3.  Spores  situated  at  end  of  rod.     Drum-stick  sporulation.  TETANUS 
GROUP. 

53 


54  STUDY   AND    IDENTIFICATION    OF    BACTERIA. 

The  following  table  taken  from  Lehmann  and  Neumann,  based 
on  pathogenic  effects,  is  of  great  practical  value.  After  inoculation 
of  some  animal  subcutaneously  with  the  suspected  material  we  have: 

A.  No  particular  symptoms  at  site  of  inoculation. 
Absorption  of  the  soluble  toxin  causing: 

(1)  General    symptoms  of  tetanus.     B.  tetani. 

(2)  Botulism  poisoning  symptoms.     Pupillary  symptoms.     Cardiac  and 
respiratory  failure. 

B.  Local  symptoms  marked  at  site  of  inoculation.     Hemorrhagic  emphysema- 
tous  oedema. 

(1)  Motile. 

(a)  Gram  negative.       B.  cedematis  maligni. 

(b)  Gram  positive.         B.  anthracis  symptomatic!. 

(2)  Nonmotile. 

B.  aerogenes  capsulatus  or  B.  phlegmonis  emphysematosae. 

SPORE-BEARING  AEROBES. 

Bacillus  Anthracis  (Pollender  discovered  1849.  Davain  re- 
cognized nature  1863.  Koch  proved  1876). — Of  the  aerobic  spore- 
bearing  bacilli  this  is  the  only  one  of  particular  medical  importance. 


FIG.  16. — Anthrax  bacilli.     Cover-glass  has  been  pressed  on  a  colony  and 
then  fixed  and  stained.     (Kolle  and  Wassermann.} 

Anthrax  is  an  important  disease  in  domestic  animals,  especially 
sheep  and  cattle.  The  characteristic  postmortem  change  in  animals 
is  the  greatly  enlarged,  friable,  mushy  spleen.  Man  is  much  less 
susceptible  than  these  animals,  but  is  more  so  than  the  goat,  horse  or 


ANTHRAX.  55 

pig.  The  Algerian  sheep  has  a  high  degree  of  immunity,  as  has  the 
white  rat.  The  brown  rat  is^quite  susceptible.  The  disease  in  man 
chiefly  occurs  among  those  working  with  hides,  wool  or  meat  of 
infected  cattle.  The  two  chief  types  in  man  are:  (i)  Malignant 
pustule  and  (2)  Woolsorter's  disease.  An  intestinal  type  is  also 
recognized.  Malignant  pustule  results  from  the  inoculation  of  an 
abrasion  or  cut;  thus  it  frequently  shows  on  the  arms  and  the  backs  of 
those  unloading  hides.  It  first  appears  as  a  pimple,  the  center  of 
which  becomes  vesicular,  then  necrotic.  A  ring  of  vesicles  surrounds 


FIG.  17. — Anthrax  bacilli  growing  in  a  chain  and  exhibiting  spores. 
(Kolle  and  Wassermann.} 

this  central  eschar  and  a  zone  of  congestion  the  vesicles.  The  lymphatics 
soon  become  inflamed  as  well  as  neighboring  glands.  The  bacilli 
are  found  especially  in  the  vesicles  or  the  lymphatics.  If  the  pustule  is 
not  excised  and  death  occurs,  there  is  not  much  enlargement  of  the 
spleen  and  the  bacteria  are  not  abundant  in  the  kidneys,  etc.,  as  with 
animals.  Man  seems  to  die  from  a  toxaemia  rather  than  a  septicaemia. 

In  woolsorter's  disease  there  is  great  swelling  and  oedema  of  the 
bronchial  and  mediastinal  glands.  The  lungs  show  oedema,  which 
about  the  bronchi  is  hemorrhagic. 

The  bacillus  is  5  to  8//  by  i  to  i  i/2fi.  It  has  square  cut  or  concave 
ends  and  is  often  found  in  chains.  It  is  Gram  positive.  Colonies,  by 
interlacing  waves  of  strings  of  bacteria,  show  Medusa  head  appearance. 


56  STUDY   AND    IDENTIFICATION    OF    BACTERIA 

For  cultural  characteristics  see  key.     Spores  develop  best  at  incubator 
temperature. 

Stiles  thinks  that  animals  are  infected  by  eating  the  bones  of 
animals  which  have  died  of  anthrax,  cutting  buccal  mucous  membrane, 
and  so  becoming  infected.  Spores  do  not  form  in  an  intact  animal 
body,  but  they  do  form  after  a  postmortem  or  the  disintegration  of 
the  body  by  maggots.  For  this  reason  it  is  better  not  to  open  up  the 
body  of  the  animal,  but  to  make  the  diagnosis  by  cutting  off  an  ear. 


FIG.  18. — Bacillus  anthracis  in  blood  of  rabbit.      (Coplin.} 
Dried  spores  will  live  for  years  and  will  withstand  boiling  temperature 
for  hours. 

In  vaccinating  animals  against  anthrax,  Pasteur  used  two  vaccines. 
The  first  is  attenuated  fifteen  days  at  42.5°  C.,  The  second,  attenuated 
for  only  ten  days,  is  given  twelve  days  later. 

In  taking  material  from  a  malignant  pustule  before  excision,  be 
careful  not  to  manipulate  it  roughly,  as  bacteria  may  enter  the  circula- 
tion. Make  cover-glass  preparations,  staining  by  Gram.  Make 
culture  on  agar.  Blood  cultures  are  usually  only  positive  later  in  the 
disease.  Inoculate  a  guinea-pig  or  a  mouse  subcutaneously. 

SPORE-BEARING  ANAEROBES. 

There  are  three  very  important  pathogens  in  this  group — that  of 
malignant  oedema;  that  of  botulism,  and  the  organism  of  tetanus. 
The  B.  enteritidis  sporogenes  is  of  importance  in  connection  with 


ANAEROBES. 


57 


indications  of  fecal  contamination  of  water.  In  connection  with  B. 
aerogenes  capsulatus,  there  is  some  question  as  to  whether  the  extensive 
oedema  produced  by  it  may  not  usually  be  from  a  terminal  or  cadaveric 
infection. 

To  Cultivate  Anaerobes. — Probably  the  apparatus  giving  the 
most  perfect  anaerobic  conditions  is  the  Novy  jar,  in  which  the  air  has 
been  replaced  by  hydrogen.  The  difficulties  attending  the  method  are : 

1.  Unless  a  special  ap- 
paratus  (Kipp's)  is 
at  hand,  there  may 
be  difficulty  in  pre- 
venting     the      sul- 
phuric    acid     from 
frothing  over  when 
poured  on  the  zinc. 
It    should,  at  first, 
be   added  in  small 
quantities  at  a  time 
— well     diluted     (i 
to  6). 

2.  Various  wash-bottles 
are    required :     one 
containing        silver 
nitrate   solution  for 
traces  of  AsH3  and 

one  with  lead  acetate  for  H2S  and  another  with  pyrogallic 
acid  and  caustic  soda  for  any  oxygen  that  may  come  over. 

3.  Mixtures  of  hydrogen  and  air  explode.     Consequently,  in 
determining  whether  all  air  has  been  expelled  and  in  its 
place  an  atmosphere  of  hydrogen  exists,  it  is  necessary  to 
see  if  the  escaping  gas  burns  with  a  blue  flame.     Unless 
this  is  collected  in  a  test-tube  and  examined,  we  may 
have  an  explosion. 

4.  Except  in  a  large  laboratory,  where  the  apparatus  is  set  up 
and  ready  for  use,  too  much  time  would  be  required. 

5.  Simpler  methods  appear  to  give  as  good  results. 


FIG.  19. — Novy  jar. 


STUDY    AND    IDENTIFICATION    OF    BACTERIA. 


The  Method  of  Liborius. — In  this  it  is  necessary  to  have  a  test- 
tube  containing  about  four  inches  of  a  one  percent  glucose  agar. 
Glucose  acts  as  a  reducing  agent  and  furnishes  energy.  It  is  conven- 
ient to  add  about  one-tenth  of  one  percent  of  sulphindigotate  of  soda ; 
the  loss  of  the  blue  color  at  the  site  of  a  colony  enabling  us  to  pick  them 

out.  The  tube  of  agar  should  be  boiled 
just  before  using  to  expel  remaining 
oxygen  from  the  tube.  Now  rapidly 
bring  down  the  temperature  to  about 
42°  C.,  by  placing  the  tube  in  cold  water, 
and  inoculate  the  material  to  be  ex- 
amined. A  second  or  third  tube  may  be 
inoculated  from  the  first,  just  as  in  ordi- 
nary diluting  methods  for  plate  cultures. 
Having  inoculated  the  tubes,  solidify 
them  as  quickly  as  possible,  using  tap- 
water  or  ice-water.  The  anaerobic 
growth  develvops  in  the  depths  of  the 
medium.  Some  pour  a  little  sterile  vaselin 
or  paraffin, or  additional  agar  on  the  top 
of  the  medium  in  the  tube  as  a  seal  from 
the  air.  Others  have  recommended  the 
inoculation  of  some  aerobe,  as  B.  prodi- 
giosus,  on  the  surface.  This  latter  method 
is  not  advisable.  A  deep  stab  culture  is 
often  sufficient. 

The  Method  of  Buchner.— In  this 
method  one  gram  each  of  pyrogallic  acid 
and  caustic  potash  or  soda  for  every 
100  c.c.  of  space  in  the  vessel  containing 
the  culture  is  used  to  absorb  the  oxygen. 

It  is  convenient  to  drop  in  the  pyrogallic  acid;  then  put  in  place  the 
inoculated  tubes  or  plates;  then  quickly  pouring  in  the  amount  of 
caustic  soda,  in  a  10%  aqueous  solution,  to  immediately  close  the  con- 
taining vessel.  A  large  test-tube  in  which  a  smaller  one  containing 
the  inoculated  medium  is  placed,  and  which  may  be  closed  by  a 


FIG.  20. — Arrangement  of 
tubes  for  cultivation  of  anae- 
robes by  Buchner's  method. 
(Williams.) 


CULTIVATION   OF  ANAEROBES.  59 

rubber  stopper,  is  very  convenient.  A  good  rubber-band  fruit  jar  is 
satisfactory.  A  desiccator  may  be  used  for  plates.  An  excellent 
method  for  anaerobic  plates,  either  in  a  desiccator  with  the  pyrogallic 
acid  and  caustic  soda,  or  less  satisfactorily  in  the  open  air,  is  to 
sterilize  the  parts  of  the  Petri  dish  inverted;  that  is,  the  smaller  part  is 
put  bottom  downward  in  the  inverted  cover  (as  one  would  set  one 
tumbler  in  another).  Then,  in  using,  unwrap  the  Petri  dish,  lift  up  the 
inner  part,  pour  in  the  inoculated  medium  into  the  upturned  cover. 
Then  immediately  press  down  the  inner  dish,  spreading  out  a  thin 
film  of  medium  between  the  two  bottoms. 

J.  H.  Wright's  Method. — Make  a  deep  stab  culture  in  glucose  agar 
or  gelatin,  preferably  boiling  the  media  before  inoculating.  Then 
flame  the  cotton  plug  and  press  it  down  into  the  tube  so  that  the  top 
lies  about  three-fourths  of  an  inch  below  the  mouth  of  the  test-tube. 
Next  fill  in  about  one-fourth  of  an  inch  with  pyrogallic  acid;  then  add 
2  or  3  c.c.  of  a  10%  solution  of  caustic  soda,  and  quickly  insert  a 
rubber  stopper.  This  method  is  one  of  the  most  convenient  and 
practical,  and  is  to  be  strongly  recommended. 

Method  of  Vignal. — In  this  a  section  of  glass  tubing  (1/4  in.)  is 
drawn  out  at  either  end,  as  in  making  a  bacteriological  pipette,  with  a 
mouth-piece  containing  a  cotton  plug.  The  liquid  agar  or  gelatin  is 
then  inoculated  and  the  medium  drawn  up  into  the  tube.  In  a  very 
small  flame  the  capillary  narrowings  are  sealed  off,  and  we  have 
inside  the  tube  very  satisfactory  anaerobic  conditions.  To  get  at  the 
colonies,  file  a  place  on  the  tube  and  break  at  this  point.* 

B.  (Edematis  Maligni  (Pasteur,  1877). — This  is  the  vibrion 
septique  of  Pasteur.  It  is  found  in  garden  soil  and  in  street  sweepings. 
It  is  the  cause  of  an  acute  cellular  necrosis  attended  with  serous  san- 
guinolent  exudation  and  with  more  or  less  emphysema.  The  organ- 
ism only  becomes  generalized  in  the  blood  about  the  time  of  death  and 
postmortem.  Therefore,  it  is  not  a  septicaemia,  as  is  anthrax.  The 
bacillus  is  an  organism  about  the  size  of  anthrax  (7//  by  .8),  but  is 
narrower  and  does  not  have  the  same  square  cut  or  dimpled  ends. 

*  To  obtain  material  for  examination  and  isolation  in  pure  culture  from  the  deep 
agar  stab-tube,  it  is  best  to  loosen  the  medium  at  the  sides  of  the  tube  with  a  heated 
platinum  spud  or  a  flattened  copper  wire.  Then  shake  the  mass  out  into  a  sterile 
Petri  dish.  It  is  dangerous  to  break  the  tubes  with  a  hammer  as  some  do. 


60  STUDY   AND    IDENTIFICATION    OF    BACTERIA. 

Furthermore,  it  is  motile,  Gram  negative  and  an  anaerobe.  The 
guinea  pig  is  very  susceptible,  and  about  the  time  of  death  and  post- 
mortem there  may  be  seen  long  flexile  motile  filaments,  15  to  40^  long, 
which  move  among  the  blood-cells  as  a  serpent  in  the  grass  (Pasteur). 

In  cultures  it  grows  out  very  slightly  from  the  line  of  stab,  giving  a 
jagged  granular  line,  differing  from  tetanus.  Spores  form  best  at  37°  C. 
— requiring  about  48  hours.  It  liquefies  gelatin.  In  examining  an 
exudate  from  a  suspected  case,  note  the  presence  of  spores  centrally 
situated.  Inoculate  a  guinea-pig.  Death  occurs  in  about  two  days. 
There  is  intense  hemorrhagic  emphysematous  oedema  at  the  site  of 


FIG.  21. — Bacillus  of  botulism.     (Kolle  and  Wassermann.} 

inoculation.  The  subcutaneous  tissue  contains  fluid  and  gas.  There 
is  present  the  foul  odor  of  an  anaerobe.  Examine  for  the  long  filaments 
showing  flowing  motility.  Be  sure  to  stain  by  Gram.  (Negative.) 
For  cultures,  heat  the  material  (either  from  a  wound  or  from  a  guinea- 
pig)  which  shows  spores  to  a  temperature  of  80°  C.  for  from  15  minutes 
to  one  hour.  Then  inoculate  glucose  agar  stab  culture  and  grow 
anaerobically.  Courmont  differentiates  anthrax  from  malignant 
oedema  by  injecting  into  ear  vein  of  rabbit.  The  injection  of  malig- 
nant cedema  in  this  way,  instead  of  subcutaneously,  tends  to  immunize. 
B.  Botulinus  (Van  Ermengem,  1896). — This  is  the  organism 
which  produces  botulism,  a  form  of  meat  poisoning.  It  is  a  spore- 


BOTULISM.  6 1 

bearing  anaerobe  and  must  not  be  confused    with  another  organism 
associated  with  meat  poisoning — the  B.  enteritidis  of  Gartner. 

There  are  dysphagia,  paralysis  of  eye-muscles  and  cardiac  and 
respiratory  symptoms  (medulla).  The  symptoms  are  due  to  the 
elaboration  of  a  soluble  toxin  of  the  same  nature  as  that  of  diphtheria 
and  tetanus.  There  is  no  fever  and  consciousness  is  preverved.  It 
has  been  isolated  from  sausage  and  ham.  It  is  a  large  bacillus — 5  to 
iQfji  x  ifjL.  It  is  motile  and  stains  by  Gram.  It  produces  gas  in 
glucose  media.  When  the  toxin  is  introduced,  it  requires  a  period  of 
incubation  of  twelve  hours.  An  important  point  is  that  ham  may  not 


FIG.  22. — Symptomatic  anthrax  (Rauschbrand)  bacilli  showing  spores. 
(Kolle  and  Wassermann.) 

appear  decomposed  and  yet  contain  many  bacilli  and  much  toxin. 
It  is  a  very  potent  toxin — as  little  as  one-thousandth  of  a  c.c.  may 
kill  a  guinea-pig.  In  man  the  toxin  is  apparently  absorbed  from  the 
alimentary  canal.  For  diagnosis  inject  an  infusion  of  the  ham  or 
sausage  which  was  eaten  of  into  a  guinea-pig,  and  characteristic 
pupillary  symptoms  with  death  by  cardiac  and  respiratory  failure  will 
result. 

Cultures  may  be  made  in  glucose  agar.  The  characteristic  point 
is  the  production  of  a  powerful  soluble  toxin  which  produces  symptoms 
when  no  bacilli  are  present. 


62  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

B.  Tetani  (Nicolaier,  1885;  Kitasato,  1889).— This  is  the  most 
important  organism  of  the  anaerobic  spore  bearers.  Its  character- 
istics are  the  tetanic  symptoms  produced  by  the  toxin  and  the  strictly 
terminal  drum-stick  spores.  Spores  are  difficult  to  find  in  material 
from  wounds  infected  with  tetanus,  but  readily  develop  in  cultures. 
Prior  to  the  formation  of  spores  the  organism  is  a  long  thin  bacillus 
(4  x  .4  ft).  It  is  motile  and  Gram  positive.  It  liquefies  gelatin  slowly 
and  does  not  coagulate  milk.  Theobald  Smith  recommends  growing 
it  in  fermentation  tubes  containing  ordinary  bouillon,  but  to  which 
a  piece  of  the  liver  or  spleen  of  a  rabbit  or  guinea-pig  has  been 


FIG.  23. — Tetanus  bacilli  showing  end  spores.     (Kolle  and  Wassermann.) 

introduced  at  the  junction  of  the  closed  arm  and  the  open  bulb.  By 
this  method  spores  develop  rapidly  in  from  twenty-four  to  thirty-six 
hours.  Sporulation  is  most  rapid  at  37°  C.  As  there  is  always  lia- 
bility to  postmortem  invasion  of  viscera  by  ordinary  saprophytes, 
Smith  recommends  that  great  care  be  taken  not  to  handle  the  animal 
roughly  in  chloroforming  and  in  pinching  off  pieces  of  the  organ  at 
autopsy.  The  animal  must  be  healthy,  and  the  tubes  to  which  the 
piece  of  tissue  is  added  must  be  proven  sterile  by  incubation.  Smith 
calls  attention  to  the  uncertainty  of  the  temperature  at  which  tetanus 
spores  are  killed.  He  shows  that  some  require  temperatures  only 
possible  with  an  autoclave.  In  view  of  the  danger  of  tetanus,  it  is 


TETANUS.  63 

advisable  to  carefully  autoclave  all  material  going  into  bacterial 
vaccines,  such  as  salt  solution,  bottles  for  holding,  etc. 

Tetanus  seems  to  grow  better  in  symbiosis  with  aerobes;  hence  a 
lacerated  dirty  wound  with  its  probable  contamination  with  various 
cocci,  etc.,  and  its  difficulty  of  sterilization,  offers  a  favorable  soil. 
The  tetanus  bacillus  gives  rise  to  one  of  the  most  powerful  poisons 
known;  it  is  a  soluble  toxin  like  diphtheria  toxin,  and  it  is  estimated 
that  1/300  of  a  grain  is  fatal  for  man. 

Rosenau  has  established  an  antitoxin  unit  for  tetanus  which  has 
the  power  of  neutralizing  one-thousand  minimal  lethal  doses.  Con- 
sequently it  is  a  unit  ten  times  as  neutralizing  as  the  diphtheria  anti- 
toxin one.  The  antitoxin  of  tetanus  is  less  efficient  than  that  of 
diphtheria  for  the  following  reasons: 

1.  There  is  about  three  times  as  great  affinity  in  vitro  between 
diphtheria  toxin  and  antitoxin  than  is  the  case  with  tetanus. 

2.  The  tetanus  toxin  has  greater  affinity  for  nerve-cells  than  for 
antitoxin. 

3.  Treatment    with    antitoxin    is    successful   after    symptoms   of 
diphtheria   appear.     With   tetanus   it   is  almost   hopeless   after   the 
disease  shows  itself.     Hence  the  importance  of  the  early  bacteriological 
examination  of  material  from  a  suspicious  wound  (rusty  nail). 

4.  The  tetanus  toxin  ascends  by  way  of  the  axis  cylinder,  and  the 
antitoxin  being  in  the  circulating  fluids  cannot  reach  it,  whereas  with 
diphtheria  both  toxin  and  antitoxin  are  in  the  circulation.     Diphtheria 
also  selects  the  cells  of  parenchymatous  and  lymphatic  organs  which 
are  more  tolerant  of  injury  than  the  nerve  cells. 

That  the  disease  is  due  to  toxin  is  shown  not  only  experimentally, 
but  also  if  spores  are  carefully  freed  of  all  toxin  by  washing,  and 
then  introduced  they  do  not  cause  tetanus — the  polymorphonuclears 
engulfing  them.  The  importance  of  the  presence  of  ordinary  pus 
cocci  in  a  tetanus  wound  may  be  that  the  activity  in  phagocytizing 
them  allows  the  tetanus  bacillus  to  escape  phagocytosis.  This  would 
also  explain  the  importance  of  necrotic  tissue  in  a  lacerated  wound — 
the  phagocytes  taking  this  up  instead  of  tetanus  bacilli.  The  toxin 
is  digested  by  the  alimentary  canal  juices  and  infection  by  that  atrium 
is  improbable.  The  infection  occurs  especially  through  skin  wounds, 


64  STUDY   AND    IDENTIFICATION    OF    BACTERIA. 

but  also  from  those  of  mucous  mem- 
brane. The  usual  period  before 
symptoms  occur  is  fifteen  days.  The 
shorter  the  period  of  incubation,  the 
more  probably  fatal  the  disease.  The 
horse  is  the  most  susceptible  animal, 
next  the  guinea  pig,  then  the  mouse. 
Fowls  are  practically  immune. 

In  examining  for  tetanus,  scrape 
out  the  material  from  the  suspected 
wound  with  a  sterile  Volkmann  spoon 
and  put  it  in  a  tube  containing  blood- 
serum.  Place  this  in  an  incubator. 
We  have  here  the  principle  of  the 
septic  tank — the  cocci  and  other 
aerobes  grow  luxuriantly  and  enable 
the  tetanus  bacillus  to  develop.  From 
day  to  day  smell  the  culture,  and  if  an 
odor  similar  to  the  penetrating,  sour, 
foul  smell  of  the  stools  of  a  man  who 
has  been  on  a  debauch  be  detected,  it 
is  suspicious.  The  nondevelopment 
of  a  foul  odor  is  against  tetanus.  Also 
make  smears  from  the  material  and 
examine  for  drum- stick  spores.  If 
these  are  found,  heat  the  material  to 
80°  C.  for  one-half  hour,  to  kill  non- 
sporing  aerobes  and  facultative  anae- 
robes, and  then  inoculate  a  deep 
glucose  agar  tube  and  cultivate  by 
Wright's  method.  The  fusiform  lateral 
outgrowth  about  the  middle  of  the 

stab  is  characteristic, 
ric.  24. — B.  aerogenes  capsulatus  ^  i 

agar  culture  showing  gas  formation.  B-     AerOgCnesCapSUlatuS 

(Welch,   1891).— This  bacillus  is  ap- 
parently widely  distributed.      It  is  possibly    the  same  organism  as 


GAS    BACILLUS.  65 

Klein's  B.  enteritidis  sporogenes,  which  is  constantly  present  in  feces. 
It  is  a  large  capsulated  organism,  which  does  not  form  chains.  Spores 
are  produced  on  blood-serum.  These  are  frequently  absent  on  other 
media.  It  is  questioned  whether  its  pathogenicity  is  other  than 
exceedingly  feeble,  the  presence  of  the  bacillus  in  emphysematous 
findings  at  postmortem  being  attributed  to  terminal  or  cadaveric 
invasion.  Cases,  however,  in  the  Philippines,  have  been  reported 
following  caribou  horn  wounds,  in  which  most  serious  and  fatal 
results  attended  emphysematous  lesions  showing  this  bacillus. 


CHAPTER  VII.^ 

STUDY    AND     IDENTIFICATION     OF    BACTERIA.     MYCO- 
BACTERIA  AND  CORYNEBACTERIA.     KEY  AND  NOTES. 

Key  for  Bacilli. — Having  branching  characteristics,  as  shown  by 
parallelism,  branching,  curving  forms,  V-shapes,  clubbing  at  ends, 
segmental  staining,  etc. 

Acid-fast.      Mycobacterium.      { 

I.  Grow  rapidly  on  ordinary  media  at  room  temperature. 

Examples:       Timothy  grass  bacillus  of  Moeller  (B.  phlei). 

Mist  bacillus.     Butter  bacilli  as  reported  by  (i)   Rabino- 
witsch  and  (2)  Petri. 

II.  Only  grow  at  about  incubator  temperature.     Scanty  growth  or  none  at  all 
on  ordinary  media.     Media  of  preference  are:  (a)  solidified  blood- serum, 
(b)  glycerin  agar  and  (c)  glycerin  potato. 

1.  Cultures  fairly  moist,  luxuriant  and  flat.     Op.  temp.  43°  C. 
a.  Bacillus  of  avian  tuberculosis. 

2.  Cultures  scanty,  wrinkled  and  dry.     Appear  in  10  to  14  days.     Op 
temp.  38°  C.     Bacilli  longer,  narrower  and  more  beaded. 

a.  Bacillus  of  human  tuberculosis. 

Cultures  as  above,  but  even  more  scanty.     Bacilli  shorter,  thicker, 
more  even  in  staining  and  more  regular  in  size. 

b.  Bovine  tubercle  bacilli. 

3.  Very  difficult  to  cultivate  (Czaplewski). 
Smegma  bacilli  of  various  animals. 

III.  Noncultivable  (at  present). 

i.  B.  leprae.  Found  chiefly  in  nasal  mucus  and  in  juice  from  lepra  tuber- 
cles. Less  often  in  nerve  leprosy. 

Nonacid-fast.     Corvnebac.eriun,  {  %?%?£*£*  ^ 

I.  Do  not  stain  by  Gram's  method. 

i.  B.  mallei  (Glanders).  Characteristic  culture  is  that  on  potato. 
Growth-like  layer  of  honey  by  third  day.  Becomes  darker  in  color, 
until  on  eighth  day  is  reddish-brown  or  opaque  with  greenish-yellow 
margin. 

II.  Gram  positive. 

1.  Very  luxuriant  growth  on  ordinary  media.     Colonies  often  yellow  to 
brownish.    B.  pseudodiphtheriae.    Shorter,  thicker  and  stain  uniformly. 

2.  Moderate  growth  on  ordinary  media.     B.  diphtherias.     Best  media  are 
blood-serum  (LofSer's)  or  glycerin  agar.     Has  metachromatic  granules 
at  poles. 

3.  Scanty  and  slow  growth  on  nutrient  media.     B.  xerosis. 

66 


TUBERCULOSIS  IN  GUINEA  PIG.  67 

THE  GROUP  OF  ACID-FAST  BRANCHING  BACILLI. 

There  is  a  large  and  ever-increasing  number  of  organisms  which 
have  the  same  staining  reactions  as  the  tubercle  bacilli,  but  which 
differ  in  four  important  essentials  of — 

1.  Growing  readily  on  any  media. 

2.  Showing  more  or  less  abundant  growth  or  colonies  in 
twenty-four  hours. 

3.  Having  no  pathogenic  power  for  guinea-pigs  when  inocu- 
lated subcutaneously. 

4.  Not  requiring  body  temperature  for  development,  but 
growing  at  room  temperature. 

Many  of  these  organisms,  if  injected  intraperitoneally  into  guinea- 
pigs,  will  produce  a  peritonitis  with  false  membrane.  Some  also 
produce  granulation  tissue  nodules  which  may  be  confused  with  true 
tubercles.  For  this  reason  it  is  well  to  study  the  lesions  in  experi- 
mental tuberculosis  in  the  guinea-pig.  Injected  subcutaneously,  on 
either  or  both  sides  of  the  posterior  abdomen  with  the  needle  pointing 
toward  the  inguinal  glands,  we  may  have  caseation  and  ulceration  at 
the  site  of  inoculation.  The  glands  in  relation  enlarge  and  caseate. 
Smears  from  these  show  T.  B.  The  marked  and  characteristic  change 
is  the  enormous  enlargement  of  the  spleen,  which  is  studded  with 
grayish  and  yellow  tubercles.  Make  smears  and  cultures  from  the 
spleen.  The  death  of  the  guinea-pig  usually  occurs  in  about  two 
months.  The  lesions  may  be  looked  for  at  three  to  five  weeks. 

These  nonpathogenic  acid-fast  bacilli  are  of  greatest  importance  by 
reason  of  their  possible  confusion  with  the  true  tubercle  bacilli. 
Their  colonies  correspond  more  or  less  with  different  types  of  tubercle 
bacilli  colonies,  being  either  dry  and  wrinkled  like  human  or  moist  and 
irregularly  flat  as  avian.  Eventually  the  moist  colonies  become  dry 
and  wrinkled.  They  have  been  isolated  from— 

1.  Butter  and  milk. 

2.  From  grasses,  especially  in  timothy  grass  infusion. 

3.  In  various  excretions  of  animals,  as  in  dung,  urine,  etc. 

4.  Normally  in  man — from  skin,  nasal  mucus,  cerumen  and 
tonsillar  exudate. 


68 


STUDY    AND    IDENTIFICATION    OF    BACTERIA. 


FIG.  25. — Bacillus  tubercu- 
losis; glycerine  agar-agar  cul- 
ture, [several  months  old. 
(Curtis.} 


It  is  important  to  remember  that  such 
organisms  have  very  rarely  been  reported 
from  pulmonary  lesions,  and  when  pres- 
ent they  have  been  considered  as  proba- 
bly causative.  Consequently  the  finding 
of  tubercle  bacilli  in  sputum  has  practi- 
cally as  great  value  as  it  had  before  we 
knew  of  these  various  acid  fast  bacteria. 

Tubercle  Bacillus  (Koch,  1882).— 
This  is  a  rather  long,  narrow  rod,  3  x.  3//.. 
In  the  human  type  it  tends  to  show  a 
beaded  appearance,  this  not  being  due  to 
spores,  however.  In  the  bovine  type  the 
staining  is  .more  solid,  the  organism 
shorter  and  thicker,  and  shows  even  a 
more  scanty  growth  than  human  T.  B. 
It  has  been  established  that  many  of  the 
tuberculous  affections  of  man,  especially 
those  of  the  skin,  bone  and  mesenteric 
glands,  are  of  the  bovine  type,  while,  as  a 
rule,  pulmonary  and  laryngeal  lesions 
are  of  the  human  type.  Experiments  by 
various  commissions  in  different  countries 
have  shown  that  human  and  bovine  types 
are  very  closely  related  and  that  not  only 
may  a  bovine  strain  affect  man,  but  that 
human  T.  B.  may  infect  young  calves. 
It  is  a  question  whether  the  avian  type  is 
absolutely  distinct;  many  experiments 
having  indicated  the  impossibility  of  in- 
fecting fowls  with  human  T.  B.  Nocard, 
by  inserting  collodion  sacs  containing 
bouillon  suspensions  of  human  T.  B., 
claims  to  have  changed  these  to  the  avian 
type.  The  avian  type  grows  at  43°  C. 
fairly  luxuriantly,  as  a  moist,  more  or 


TUBERCULINS.  69 

less  spreading  culture.  There  is  also  a  fish  tuberculosis.  This 
organism  grows  much  more  rapidly  than  the  other  types  (3  to  4  days), 
and  grows  best  at  24°  C.,  growth  ceasing  at  36°  C.  The  colonies  are 
round  and  moist. 

The  best  culture  media  for  primary  cultures  are  blood-serum  or, 
better,  a  mixture  of  yolk  of  egg  and  glycerin  agar.  Dorset's  egg 
medium  is  also  used.  In  subcultures,  either  glycerin  agar,  glycerin 
potato  or  glycerin  bouillon  make  good  media.  In  inoculating  media 
from  tuberculous  material,  as,  say,  from  a  tuberculous  gland  or,  more 
practically,  from  the  spleen  of  a  guinea-pig,  the  material  must  be 
thoroughly  disintegrated  or  rubbed  on  the  surface  of  the  media  so 
that  individual  bacilli  may  rest  on  the  surface  of  the  culture  media. 
In  growing  in  flasks  in  glycerin  bouillon  a  surface  growth  is  desired. 
The  cylindrical  flask  of  Koch  gives  a  better  support  to  the  pellicle  than 
an  Erlenmeyer  one.  In  inoculating,  a  scale  of  such  a  surface  growth 
or  a  grain  from  the  growth  on  a  slant  should  be  deposited  on  the 
surface  of  the  glycerin  bouillon  in  the  flask. 

Inasmuch  as  the  filtrate  from  cultures  has  little  toxic  effect,  the 
poison  is  assumed  to  be  intracellular. 

Koch's  old  tuberculin,  which  wras  simply  a  concentrated  glycerin 
bouillon  culture  killed  by  heat,  is  now  principally  used  in  veterinary 
diagnosis. 

Koch's  tuberculin  "R"  or  new  tuberculin  was  introduced  in  1897. 
In  this,  virulent  bacilli  are  dried  In  vacuo,  ground  up  in  water  and 
centrifuged.  The  first  supernatant  fluid  is  put  aside  as  tuberculin  O. 
Subsequent  trituration  and  centrifugalization,  preserving  each  time 
the  supernatent  suspension,  gives  the  new  tuberculin.  It  has  been 
found  at  times  to  contain  virulent  T.  B. 

Koch's  bazillen  emulsion  has  been  recently  introduced  by  Koch. 
This  is  simply  a  suspension  of  ground-up  bacilli  in  50%  glycerin  solu- 
tion. It  contains  both  T.  R.  and  T.  O. 

In  the  use  of  T.  R.  and  of  bazillen  emulsion,  Sir  A.  Wright  recom- 
mends doses  of  1/4000  of  a  milligram,  and  he  rarely  goes  beyond 
i/ 1000  of  a  milligram  in  treatment.  These  products  come  in  i  c.c. 
bottles  containing  10  mgs.  of  bacillary  material.  It  is  convenient  to 
remove  i/io  of  a  c.c.,  containing  i  mg.  Add  this  to  10  c.c.  of  glycerin 


70  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

salt  solution  with  1/4%  of  lysol.  Each  c.c.  contains  i/io  mg.  One 
c.c.  of  this  stock  solution  added  to  99  c.c.  of  salt  solution,  with  1/4% 
of  lysol,  would  give  a  working  solution,  each  c.c.  of  which  would 
contain  i/iooo  mg.  of  tuberculin. 

For  diagnostic  reactions  we  have,  besides  the  method  of  injecting 
tuberculin  and  noting  presence  or  absence  of  fever,  four  more  recent 
diagnostic  tuberculin  tests  :  (i)  Variations  in  opsonic  index.  (2) 
Instillation  into  one  eye  of  a  drop  of  1/2%  or  i%  -solution  of  purified 
tuberculin.  Reaction  is  shown  by  redness,  especially  of  inner  canthus, 
in  12  to  24  hours  (Calmette).  (3)  The  cutaneous  inoculation 
method  similar  to  ordinary  vaccination  methods.  The  appearance  of 
bright  red  papules  in  24  hours  indicates  reaction  (von  Pirquet). 
(4)  Ointment  tuberculin  test.  Rub  in  50%  ointment  of  tuberculin  in 
lanolin.  Reaction  is  shown  by  dermatitis  with  reddened  papules  in 
24  to  48  hours.  (5)  Inoculation  of  bovine  and  human  tuberculin  to 
diagnose  type  of  infection  (Detre). 

In  staining  it  is  better  to  use  the  Ziehl-Neelsen  method,  decolor- 
izing with  3%  hydrochloric  acid  in  95%  alcohol.  The  alcohol,  for  all 
practical  purposes,  enables  us  to  eliminate  the  smegma  and  similar 
bacilli,  these  being  decolorized  by  such  treatment.  There  are  two 
objections  to  the  Gabbett  method,  where  decolorizer  and  counter- 
stain  are  combined:  (i)  We  cannot  judge  of  the  degree  of  decolor- 
ization — we  are  working  in  the  dark;  and  (2)  the  matter  of  elimination 
of  smegma  bacilli  is  impossible. 

Pappenheim's  method,  in  which  corallin  and  methylene  blue  are 
dissolved  in  alcohol,  does  not  appear  to  have  any  advantage  over  acid 
alcohol.  As  a  practical  point  when  the  question  of  tuberculosis  of 
the  genito-urinary  tract  is  involved,  inoculate  a  guinea-pig  with 
urinary  sediment. 

It  must  be  remembered  that  in  young  cultures  of  tubercle  bacilli 
many  of  the  rods  are  nonacid-fast,  taking  the  blue  of  the  counter- 
stain,  while  older  rods  are  acid-fast.  This  frequently  causes  suspicion 
of  a  contaminated  culture. 

Bacillus  Leprae  (Hansen,  1874).— This  is  the  cause  of  leprosy. 
In  nodular  leprosy  the  organism  is  readily  and  in  the  greatest  abun- 
dance found  in  the  juice  of  the  tubercles  of  the  skin,  nasal  and  pharyn- 


LEPROSY.  71 

geal  mucosa.  In  the  skin  they  are  chiefly  found  in  the  derma,  espe- 
cially in  connective-tissue  cells  and  endothelial  cells.  They  are  also 
found  in  the  glands  in  relation  to  the  superficial  lesions.  The  bacilli 
are  found  in  smaller  numbers  in  the  liver  and  spleen.  In  anaesthetic 
or  nerve  leprosy  they  are  found  in  small  numbers  in  the  granuloma 
tissue  which  affects  the  interstitial  connective  tissue  of  the  peripheral 
nerves.  Also,  rarely,  in  the  anaesthetic  spots  of  nerve  leprosy. 

In  morphology  and  staining  reactions  they  are  almost  identical 
with  the  tubercle  bacillus.  The  main  points  of  distinction  are:  (i) 
The  fact  of  the  leprosy  bacilli  being  found  in  enormous  numbers, 
especially  in  large  vacuolated  cells  (lepra  cells),  and  lying  in  the  lymph 
spaces.  They  are  frequently  beaded  and  lie  in  masses  which  have  been 
likened  to  a  bundle  of  cigars  tied  together.  It  is  necessary  to  examine 
for  long  periods  of  time,  smears  made  from  tuberculous  lesions  of 
skin  before  finding  a  single  organism.  (2)  Leprosy  bacilli  have  not 
surely  been  cultivated.  (3)  Injected  into  guinea-pigs,  they  do  not 
produce  lesions. 

Recently  a  leprosy -like  disease  of  rats  has  been  reported  in  which 
there  are  two  types:  (i)  A  skin  affection  and  (2)  a  glandular  one. 
In  this  disease  acid-fast  bacilli,  alike  in  all  respects  to  leprosy  bacilli, 
have  been  found.  Deane  has  obtained  a  diphtheroid-like  organism 
in  culture,  which  is  nonacid-fast.  This  same  finding  has  been 
obtained  in  cultures  considered  positive  in  human  leprosy.  Quite 
recently  it  has  been  claimed  that  the  bacillus  has  been  cultivated  by 
excising  aseptically  the  subcutaneous  portion  of  lepromata  and 
dropping  the  leprous  tissue  into  salt  solution. 

For  diagnosis  we  should  use  both  smears  from  the  nasal  mucus 
and  from  ulcerated  lepromata  or  from  the  scrapings  from  intact 
tubercles.  Some  advise  centrifuging  with  salt  solution,  but  this  is 
rarely  necessary.  The  best  method  is  to  excise  a  small  portion  of  skin 
or  mucous  membrane,  fix  it  in  absolute  alcohol  or  Zenker's  fluid.  Cut 
thin  sections  in  paraffin.  Stain  with  carbol-fuchsin,  decolorize  with 
acid  alcohol,  and  then  stain  with  haematoxylin.  This  gives  the  location 
of  the  bacilli.  This  is  also  a  good  method  for  tuberculous  tissues. 
It  is  claimed  that  the  B.  leprae  stains  more  easily  and  loses  its  color 
more  rapidly  than  the  tubercle  bacillus.  Some  prefer  to  stain  the 


72  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

leprosy  bacillus  by  Gram's  method,  it  as  well  as  the  tubercle  bacillus 
being  Gram  positive. 

NONACID-FAST   BRANCHING    BACILLI. 

Bacillus  Mallei  (Lofifler  and  Shutz,  1882). — This  is  the  cause  of  a 
rather  common  disease  of  horses.  When  affecting  the  superficial 
lymphatic  glands,  it  is  termed  "farcy;"  when  producing  ulceration 
of  nasal  mucous  membrane,  the  term  "glanders"  is  used. 

In  man  there  are  two  types  of  glanders — chronic  and  acute.  In  the 
chronic  form  an  abrasion  becomes  infected  from  contact  with  glanders 
material  and  an  intractable  foul  discharging  ulceration  results.  This 
may  persist  for  months  with  lymphatic  involvement  or  may  become 
acute.  The  acute  form  may  also  develop  from  the  start  and  the  cases 
are  usually  diagnosed  as  pyaemia.  Death  invariably  results  in  acute 
glanders.  The  bacillus  is  a  narrow,  slightly  curved  rod,  about  3  x  .^IJL. 
It  is  nonmotile  and  Gram  negative.  It  at  times  presents  a  beaded 
appearance.  In  subculture  on  agar  or  blood-serum  the  growth  is 
somewhat  like  typhoid,  but  more  translucent.  In  original  cultures 
from  pus  or  tissues  the  colonies  may  not  show  themselves  for  48  hours. 
The  characteristic  culture  is  that  on  potato.  Grown  at  37°  C.,  we  have 
a  light  brown  mucilaginous  growth,  which  by  the  end  of  a  week  spreads 
out  and  takes  a  cafe  au  lait  color.  The  potato  assumes  a  dirty-brown 
color.  This  and  the  inoculation  of  a  guinea-pig  are  the  chief  diag- 
nostic measures.  If  the  material  is  injected  intraperitoneally  into  a 
male  guinea-pig,  marked  swelling  of  the  testicles  is  noted  within  48 
hours. 

The  best  stains  are  carbol  thionin  and  formol  fuchsin.  In  sections 
stained  with  carbol  thionin  the  bacilli  are  apt  to  be  decolorized  by  the 
subsequent  passage  of  the  section  through  alcohol  and  xylol.  This 
may  be  avoided  by  blotting  carefully  after  the  thionin,  then  clearing 
with  xylol  or  some  oil,  and  mounting.  Nicolle's  tannin  method  is  a 
good  one. 

Mallein  is  prepared  by  sterilizing  cultures  that  have  grown  in 
glycerin  bouillon  for  about  a  month  by  means  of  heat  (100°  C.). 
The  dead  culture  is  then  filtered  through  a  Berkefeld  filter  and  the 
filtrate  constitutes  mallein.  It  is  chiefly  used  as  a  means  of  diagnosing 


DIPHTHERIA.  73 

the  disease  in  horses.     The  reaction  consists  in  rise  of  temperature 
and  local  oedema.     The  dose  is  about  i  c.c. 

Bacillus  Diphtheriae  (Klebs  discovered,  1883;  Loffler  cultivated, 
1884). — The  diphtheria  bacillus  is  found  not  only  in  the  false  mem- 
brane which  is  so  characteristic  of  the  disease,  but  may  be  found  in 
abundance  in  the  more  or  less  abundant  secretions  of  nose  and  pharynx. 
In  studying  the  epidemiology  of  diphtheria,  especial  attention  must  be 
gh'en  to  the  examination  of  nasal  discharges.  The  B.  diphtheria?  may 
be  in  pure  culture  lying  entangled  in  the  fibrin  meshes  or  contained 


FIG.  26.— Bacillus  of  diphtheria.     (X  1000.)     (Williams.') 

within  leukocytes  in  the  membrane  or  be  associated  with  staphylococci, 
pneumococci  or  especially  streptococci.  These  latter  complicate 
unfavorably  and  cause  the  suppurative  conditions  about  the  neck.  In 
fatal  cases  the  diphtheria  bacillus  may  be  found  in  the  lungs.  Ordi- 
narily, however,  it  remains  entirely  local  and  does  not  get  into  the  circu- 
lation or  viscera. 

It  produces  soluble  absorbable  poisons  which  are  designated  toxin 
in  the  case  of  the  one  responsible  for  the  acute  intoxication,  parenchy- 
matous  degeneration  and  death;  and  toxone  for  the  poison  which  pro- 


74  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

duces  oedema  at  the  site  of  inoculation  and  postdiphtheritic  palsy. 
The  injection  of  the  soluble  poisons  alone  without  the  bacilli  produce 
the  symptoms  of  the  disease. 

The  bacilli  tend  to  appear  as  slighlty  curved  rods,  showing  varying 
irregularities  in  staining,  as  banding  or  beading,  and  in  particular  the 
presence  at  either  end  of  small,  deeply  staining  dots  (metachromatic 
granules).  These  are  well  seen  with  Loffler's  blue,  but  better  with 
Neisser's  method.  In  culture-  they  also  show  swelling  at  one  or  both 
ends  or  clubbing.  In  secretions  or  in  culture  they  show  V-shapes  or 
false  branching  and,  what  is  most  characteristic,  the  parallelism — four 


FIG.  27. — Diphtheria  bacilli  involution  forms.     (Kotte  and  Wassermann.} 

or  five  bacilli  lying  side  by  side  like  palisades.  Being  a  Gram  positive 
organism  while  the  majority  of  the  other  pathogenic  bacteria  are  Gram 
negative,  it  is  of  greatest  importance  to  stain  smears  by  this  method. 
It  is  not  so  strongly  tenacious  of  the  gentian  violet  as  the  cocci,  so 
decolorization  should  not  be  carried  too  far. 

The  best  medium  for  growing  it  is  Loffler's  blood-serum.  Coagu- 
lated white  of  egg  answers  equally  well,  as  will  a  hard-boiled  egg — the 
shell  at  one  end  being  cracked  and  the  white  cut  with  a  sterile  knife. 
This  smooth  side  is  then  inoculated  and  the  egg  placed  cut  side  down- 
ward in  a  sherry  glass.  If  an  incubator  is  not  at  hand  a  tube  may  be 
carried  next  the  body  in  a  pocket.  The  bacillus  grows  better  on  glycerin 


DIPHTHERIA.  75 

agar  than  on  plain  agar.  On  such  plates  they  appear  as  small,  coarsely 
granular  colonies  with  a  central  dark  area.  In  size  the  colonies 
resemble  the  streptococcus.  On  blood-serum  the  colonies  are  larger — 
1 1 12  to  1/8  inch  in  diameter.  In  bouillon  it  tends  to  form  a  surface 
growth.  It  is  at  the  surface  that  the  toxin  function  is  most  marked, 
hence  in  growing  diphtheria  for  toxin  formation  we  use  Fernbach 
flasks  which  expose  a  large  surface  to  the  air.  It  is  a  marked  acid 
producer — bouillon  with  a  +  i  reaction  becoming  +2.5  to  +3  in  36 
hours.  The  nitrate  from  a  twro-  or  three-weeks-old  broth  culture  is 
highly  toxic,  and  is  usually  referred  to  as  diphtheria  toxin.  It  is  used 
in  injecting  horses  to  produce  antitoxin.  Ehrlich  uses  as  a  standard  to 
measure  the  toxicity  of  toxin  the  minimal  lethal  dose  (M.  L.  D.)  This 
is  the  amount  of  toxin  which  will  kill  a  350  gram  guinea-pig  in  just  four 
days.  Some  toxins  have  been  produced  whose  M.  L.  D.  was  1/500 
c.c.,  so  that  one  c.c.  of  such  toxin  would  kill  500  guinea-pigs.)^  Theoreti- 
cally, the  measure  of  an  antitoxin  unit  is  the  capacity  of  neutralizing 
200  units  of  a  pure  toxin.^(  (On  exposure  to  light,  etc.,  toxin  loses  its 
toxic  power  and  is  termed  toxoid.)  Inasmuch,  however,  as  toxone 
and  toxoid  are  also  present,  we  may  practically  consider  an  antitoxin, 
or  immunizing  unit  (i.e.,  Immunitatseinheit),  as  about  capable  of 
making  innocuous  100  M.  L.  D. 

In  obtaining  material  from  a  throat,  be  sure  that  an  antiseptic 
gargle  has  not  been  used  just  prior  to  taking  the  throat  swab.  The 
part  of  the  swab  which  touched  the  membrane  or  suspicious  spot 
should  come  in  contact  with  the  serum  slant.  This  is  best  accom- 
plished by  revolving  the  swab.  An  immediate  diagnosis  is  possible  in 
probably  35%  of  cases  by  making  a  smear  from  a  piece  of  membrane. 
In  doing  this  Neisser's  stain  is  the  most  satisfactory. 

If  there  is  any  doubt  about  the  nature  of  an  organism  in  a  throat 
culture,  always  stain:  (i)  with  Loffler's  alkaline  methylene  blue  for  two 
minutes;  (2)  with  Gram's  method,  being  careful  not  to  carry  the 
decolorization  too  far,  and  (3)  by  Neisser's  method.  With  Loffler's 
you  obtain  a  picture  which,  after  a  little  experience,  is  characteristic ; 
at  times  the  polar  bodies  show  as  intense  blue  spots  in  the  lighter  blue 
bacillus.  One  is  liable  to  confuse  cocci  lying  side  by  side  for  diphtheria 
bacilli  with  segmental  or  banded  staining.  This  difficulty  is  not  ap- 


76  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

parent  when  Gram's  staining  is  used.  This  gives  us  great  information, 
as  the  diphtheria  and  the  pseudodiphtheria  are  the  only  small  Gram 
positive  bacilli  usually  found  in  the  mouth.  The  cocci  are  also  well 
brought  out.  Neisser's  stain  gives  a  picture  which,  when  satisfactory,  is 
almost  absolutely  characteristic.  You  have  the  bright  blue  dots  lying 
at  either  end  of  the  light  brownish-yellow  rods.  When  first  isolated 
from  a  throat,  the  diphtheria  bacillus  is  apt  to  stain  characteristically  by 
Neisser.  Later  on,  in  subculture,  there  may  be  no  staining  of  the 
polar  bodies.  Neisser  originally  recommended  five  seconds'  applica- 
tion, with  an  intermediate  washing,  for  each  of  his  two  solutions. 


FIG.  28. — B.  diphtheria  stained  by  Neisser's  method.     (Williams.) 

Thirty  seconds  for  each  is  probably  preferable.  Some  authorities 
recommend  five  to  thirty  minutes.  It  is  well  to  bear  in  mind  that 
about  2%  of  the  people  in  apparent  health  carry  diphtheria  bacilli  in 
their  throats. 

Diphtheroid  Bacilli.  Pseudodiphtheria  Bacillus.  Hofman's 
Bacillus. — Under  these  terms  various  Gram  positive  bacilli  have 
been  described  as  occuring  in  nose  and  in  skin  diseases: 

i.  They  very  rarely  give  the  blue  dot  staining  at  the  two  ends. 
Exceptionally  they  may  give  a  dot  at  one  end.     Neisser 


DII'HTHEROID    ORGANISMS.  77 

attaches  importance  to  the  dots  at  both  ends  as  showing 
diphtheria. 

2.  They  tend  to  stain  solidly  or  at  most  with  only  a  single 
unstained  segment.     They  are  shorter,  thicker  and  do  not 
curve  so  gracefully  as  the  true  diphtheria  bacillus.     They 
are  stockier. 

3.  They  produce  very  little  acid  in  sugar  media,  not  one-half 
that  produced  by  true  diphtheria. 

4.  They  are  nonpathogenic  for  guinea-pigs. 

5.  Many  of  them  grow  quite  luxuriantly  and  often  show 
chromogenic  power. 

Xerosis  Bacillus. — This  organism  is  frequently  found  in  normal 
conjunctival  discharges.  There  is  question  as  to  its  pathogenesis,  and 
the  finding  of  this  organism  should  not  exclude  the  previous  presence  of 
strictly  pathogenic  organisms,  such  as  the  gonococcus  or  the  Koch- 
Weeks.  It  resembles  the  diphtheria  bacillus  in  being  Gram  positive 
and  showing  parallelism,  but  differs  (i)  in  being  nonvirulent  for 
guinea-pigs;  (2)  in  requiring  about  two  days  for  the  appearance  of 
colonies;  (3)  in  not  showing  Neisser's  granule  staining,  and  (4)  in 
producing  very  little  acid  in  sugar  media. 


CHAPTER  VIII. 

STUDY  AND  IDENTIFICATION  OF  BACTERIA.      GRAM 
NEGATIVE  BACILLI.    KEY  AND  NOTES. 

KEY  to  the  recognition  of  nonspore-bearing,  nonchromogenic, 
non-Gram  staining,  nonbranching  bacilli. 

(NOTE. — Some  books  say  that  the  proteus  group  is  Gram  positive. 
It  is,  however,  usually  negative.) 

Do  not  grow  on  ordinary  media.  Require  blood  or  serum  agar  (hemophilic 
bacteria).  Minute  dew-drop  colonies. 

1.  Influenza  bacillus. 

2.  Koch- Weeks  bacillus  (conjunctivitis).     Serum  agar  best  medium. 

3.  Muller's  bacillus  of  trachoma.     Like  Koch- Weeks  bacillus,  but  easier 
to  cultivate. 

4.  Morax  diplobacillus  of  conjunctivitis.     Produces  little  pits  of  liquefac- 
tion in  serum. 

5.  Pseudoinfluenza  bacillus  (whooping-cough?). 

6.  Ducrey's  bacillus  (soft  chancre).     Requires  almost  pure  blood. 
Grow  well  on  ordinary  media. 

I.  Cultures  in  litmus  milk.     PINK. 

A.  Nonmotile. 

Lactis  aerogenes  group.     B.  lactis  aerogenes. 

Produce  gas  in  glucose    lactose,  or  saccharose.     No  liquefaction  of 

gelatin.     Short,  stubby  bacteria. 

B.  Motile. 

1.  Nonliquef action  of  gelatin. 

a.  B.  coli  group.  Coagulation  of  milk.  No  subsequent  pep- 
tonization.  Gas  in  glucose  and  lactose,  none  in  saccharose. 
Indol  produced.  Neutral  red  reduced. 

2.  Liquefaction  of  gelatin. 

a.  B.  cloacae  group.  Gas  in  glucose,  slight  in  lactose.  Slow 
coagulation  of  milk.  Subsequent  peptonization. 

II.  Cultures  in  litmus  milk.     LILAC. 

A.  Nonmotile  bacilli. 

i.  No  gas  generated  in  glucose  or  lactose  bouillon. 

a.  Hemorrhagic  septicaemia  group.     These  are  oval  bacilli  with 
tendency    to    bipolar    staining.     Examples:     B.    pestis,    B. 
suisepticus,  B.  cholera?  gallinarum  (chicken  cholera). 

b.  Dysentery   gioup.      Divided    into   two  classes  according  as 
mannite  is  acted  on: 

Those    not    giving    acid — nonacid     group — (Shiga-Kruse) . 
Those  giving  acid— acid  group — (Flexner-Strong). 

78 


INFLUENZA.  79 

2.  Gas  generated  in  glucose  bouillon  not  in  lactose. 

a.  Friedlander  group.     Give  very  viscid,  porcelain-like  colonies. 
Tendency  to  capsule  formation  in  favorable  media. 
Examples:     B.     pneumoniae,    B.    capsulatus    mucosus,    B. 
rhinoscleromatis. 
P..    Motile  burilli. 

1.  Do  not  liquefy  gelatin. 

a.  DQ  not  produce  gas  in  either  glucose  or  lactose  bouillon. 
Typhoid,  or  Eberth  group.     No  imlol.     No  coagulation  of 
milk. 

No  reduction  of  neutral  red. 

b.  Gartner  group.     This  includes: 

Pathogenic  types  for  man;  as,  B.  enteritidis,  B.  icteroides, 
B.  paratyphoid  B.  B.  psittacosis.  Nonpathogenic  for  man; 
as  B.  cholerae  suum  (hog  cholera). 

2.  Liquefy  gelatin. 

a.  Proteus  group.     Colonies  amoeboid,  spreading.     Produce  gas 
in  glucose,  not  in  lactose. 

GRAM  NEGATIVE  BACILLI  REQUIRING  SPECIAL  MEDIA. 

Bacillus  Influenzas  (Pfeiffer,  1892). — This  organism  is  the  type  of 
the  so-called  haemophilic  bacteria — organisms  whose  growth  is  re- 
stricted to  media  containing  haemoglobin.  The  influenza  bacillus 
seems  to  grow  better  on  slants  freshly  streaked  with  blood  than  on 
those  which  have  been  made  for  some  time,  and  they  appear  to  grow 
better  on  this  surface  smear  of  blood  than  on  a  mixture  of  agar  and 
blood. 

The  influenza  bacilli  are  most  likely  to  be  isolated  from  the  sputum 
of  bronchopneumonia  due  to  this  organism.  It  has  also  frequently 
been  found  in  the  nasal  secretions  of  influenza  patients.  Exception- 
ally, it  is  present  in  the  blood,  and  has  been  isolated  in  cases  of  menin- 
gitis. It  is  a  very  small  bacillus  which  tends  to  show  itself  in  aggre- 
gations, especially  centering  about  M.  tetragenus.  It  stains  rather 
faintly  when  compared  with  cocci,  so  that  a  smear  of  sputum  stained 
with  formol  fuchsin  shows  a  deep  violet  staining  for  the  M.  tetragenus 
or  other  cocci,  and  scattered  around  in  a  clump  like  aggregation  we  see 
these  minute,  rather  faintly  stained  rods.  They  also  tend  to  stain 
more  deeply  at  either  end  so  that  they  sometimes  appear  as  diplococci. 
Gram's  method,  counterstaining  with  formol  fuchsin,  is  excellent  for 
their  demonstration.  The  red  baclli  and  the  violet  black  cocci  are 
easily  distinguished. 

To  cultivate  them,  rub  the  sputum  or  the  material  from  a  lung  on  a 


8o 


STUDY    AND    IDENTIFICATION    OF    BACTERIA. 


slant  smeared  with  human  blood  (pigeon's  blood  is  also  satisfactory), 
and  then  without  sterilizing  the  loop,  innoculate  a  second  blood  slant; 
then  a  third,  and  possibly  a  fourth.  The  colonies  appear  as  very 
minute  dew  drop-like  points  which  seem  to  run  into  each  other  in  a 
wave-like  way.  Lord  distinguishes  them  from  pneumococcus  colonies 
by  their  disappearing  from  view  as  you  shift  from  reflected  light  to 
direct  transmitted  light.  To  test  such  colonies  we  should  transfer  a 
single  colony  to  plain  agar  and  blood-serum,  trying  not  to  carry  over 
any  blood.  If  the  least  trace  of  blood  is  carried  over,  they  may  grow 
on  agar  or  blood  serum.  Organisms  resembling  the  influenza  bacillus 


FIG.  29.— The  Koch- Weeks  Bacillus.     (Hansett  and  Sweet.) 


have  been  isolated  from  whooping-cough.  Such  organisms  have 
also  been  found  in  the  fauces  of  well  persons.  In  many  epidemics 
of  influenza  the  bacillus  has  not  been  isolated,  or  success  has  obtained 
in  only  a  small  proportion  of  the  cases.  The  influenza  bacillus  seems 
to  grow  best  in  symbiosis  with  some  other  organism,  especially  with  S. 
pyogenes  aureus. 

Koch-Weeks  Bacillus  (Koch,  1883). — This  produces  a  severe 
conjunctivitis.  It  is  very  common  in  Egypt  and  is  also  a  frequent 
cause  of  conjunctivitis  in  the  Philippines  and  in  temperate  climates. 

Smears  made  from  conjunctival  secretion  show  large  numbers  of 
small  Gram  negative  bacilli,  especially  contained  within  pus  cells, 


FRIEDLANDER'S  BACILLUS.  81 

but  also  lying  free.  They  are  more  difficult  to  cultivate  than  the 
influenza  bacillus,  but  the  same  general  methods  hold. 

Diplobacillus  of  Morax. — This  organism  causes  mild  conjunc- 
tivitis chiefly  at  the  internal  angle  of  the  eye.  They  are  about  i  or  2  // 
long  and  tend  to  occur  in  pairs  or  short  chains.  Some  claim  that 
they  are  Gram  positive.  They  are  haemophilic. 

Bacillus  of  Chancroid  (Ducrey,  1889).— These  are  short  cocco- 
bacilli,  occurring  chiefly  in  chains.  They  show  bipolar  staining. 
They  grow  best  in  a  mixture  of  blood  and  bouillon. 

GRAM  NEGATIVE  BACILLI  GROWING  ON  ORDINARY  MEDIA. 

Bacillus  Pneumoniae  (Friedlander,  1882). — This  organism  is 
responsible  for  about  5%  of  the  cases  of  pneumonia.  It  is  usually 
termed  the  pneumobacillus  to  distinguish  it  from  the  pneumococcus; 
at  other  times  Friedlander's  bacillus.  The  name  of  Fraenkel  attaches 
to  the  pneumococcus.  Morphologically,  it  is  a  short,  thick  bacillus, 
and  in  pathological  material,  as  sputum,  shows  a  wide  capsule.  -It  is 
nonmotile  and  Gram  negative.  The  colonies  on  agar  are  of  a  pearly 
whiteness  and  are  markedly  visci'd.' '  On  potato  it  shows  a  thick  viscid 
growth  containing  gas  bubble^.  The  characteristic  culture  is  the  nail 
culture  of  a  gelatin  stab.  The  growth  at  the  surface  is  heaped  up  like  a 
round-headed  nail,  the  line  of  puncture  resembling  the  shaft  of  the 
nail.  It  does  not  liquefy  gelatin.  It  does  not  produce  indol,  and 
does  not  produce  gas  in  lactose  bouillon — differences  from  the  colon 
bacillus — with  which  it  may  be  confused  in  cultures,  as  it  does  not 
then  possess  a  capsule.  If  in  doubt,  inject  a  mouse  at  the  root  of  the 
tail.  Death  from  septicaemia  occurs  in  two  days.  The  peritoneum  is 
sticky  and  numerous  capsulated  bacilli  are  present  in  the  blood  and 
organs.  The  organisms  which  have  been  isolated  from  rhinoscleroma 
and  ozcena  are  practically  identical  with  the  B.  pneumonias.  This 
group  of  organisms  are  generally  referred  to  as  the  Friedlander  group. 
Similar  organisms  have  been  isolated  from  the  discharges  of  middle- 
ear  diseases  and  in  anginas.  Cases  have  been  reported  where  the 
B.  pneumoniae  was  the  cause  of  septicaemia  in  man. 

Bacillus  Pestis  (Kitasato,  Yersin,  1894). — This  is  the  organism  of 
plague.  It  is  the  member  of  the  group  of  hemorrhagic  septicaemias 


82 


STUDY    AND    IDENTIFICATION    OF    BACTERIA. 


(Pasteurelloses),  from  which  man  suffers.  It  is  primarily  a  disease  of 
rats.  Other  Pasteurelloses  are  chicken  cholera  and  swine  plague. 
Where  the  plague  bacilli  are  found  chiefly  in  the  glands,  we  have 
bubonic  plague;  when  in  the  lungs,  pneumonic  plague,  and  when  as  a 
septicaemia,  septicaemic  plague.  An  intestinal  type  is  recognized  by 
c-ome  authors.  It  must  be  remembered  that  in  all  forms  of  plague  the 
lymphatic  glands  show  hemorrhagic  oedema;  it  is  in  bubonic  plague, 
however,  that  the  areas  of  necrosis  with  periglandular  oedema  are 
prominent.  Where  the  symptoms  are  slight,  mainly  buboes,  the  term 
pestis  minor  is  sometimes  used;  the  typical  disease  being  termed 
pestis  major.  In  pneumonic  plague  we  have  a  bronchopneumonia. 


FIG.  30. — Colonies  of  plague  bacilli  forty-eight  hours  old. 
(Kolle  and  Wassermann.} 

In  smears  from  material  from  buboes,  from  sputum  or  in  blood 
smears,  as  well  as  from  blood  or  spleen  smears  from  experimental 
animals,  we  obtain  the  typical  morphology  of  a  coccobacillus  1.5 
x  .$fj.  with  very  characteristic  bipolar  staining;  there  being  an  interme- 
diate, unstained  area.  Inoculating  tubes  of  plain  agar  and  3%  salt  agar 
with  this  same  material,  wre  obtain  in  plain  agar  cultures  organisms  which 
are  typically  small,  fairly  slender  rods,  which  do  not  stain  characteris- 
tically at  each  end  and  are  not  oval.  The  smear  obtained  from  the 
salt  agar  presents  most  remarkable  involution  forms — coccoid;  root- 
shaped,  sausage  shaped  forms,  ranging  from  three  to  twelve  microns 


PLAGUE  83 

in  length,  more  resembling  cultures  of  moulds  than  bacteria.  Another 
point  is  that  on  the  inoculated  plain  agar  we  are  in  doubt  at  the  end  of 
twenty-four  hours  whether  the  dew  drop-like  colonies  are  really  bac- 
terial colonies  or  only  condensation  particles.  By  the  second  day, 
however,  these  colonies  have  an  opaque  grayish  appearance,  so  that 
now,  instead  of  questioning  the  presence  of  a  culture,  we  consider  the 
possibility  of  contamination. 

The  plague  bacillus  grows  well  at  room  temperature — its  optimum 
temperature  being  30°  instead  of  37°  C.,  as  is  usual  with  pathogens. 
Next  to  the  salt  agar  culture,  the  most  characteristic  one  is  the  stalactite 


FIG.  31. — Pest  bacilli  from  spleen  of  rat.     (Kolle  and  Wassermann.) 

growth  in  bouillon  containing  oil  drops  on  its  surface.  The  culture 
grows  downward  from  the  under  surface  of  the  oil  drops  as  a  powdery 
thread.  These  are  very  fragile,  and  as  the  slightest  jar  breaks  them,  it 
is  difficult  to  obtain  this  cultural  characteristic. 

In  diagnosing  always  use  animal  experimentation.  Albrecht  and 
Ghon  have  shown  that  by  smearing  material  into  the  intact,  shaven 
skin  of  a  guinea-pig,  infection  occurs.  This  is  the  most  crucial  test. 
Mice  inoculated  at  the  root  of  the  .tail  quickly  succumb.  Rats,  this 
being  a  primary  disease  of  rats,  are  of  course  susceptible.  In  natural 
plague  of  rats,  the  lesions,  which  establish  a  diagnosis  even  without  the 
aid  of  a  microscope,  are  subcutaneous  injection  of  the  flaps  of  the 


84  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

abdominal  walls  as  they  are  turned  back,  fluid  in  the  pleural  cavities, 
ccdematous  haemorrhagic  swelling  of  the  neck  glands,  and  in  particu- 
lar a  creamy,  mottled  appearance  of  the  liver.  Smears  from  the  spleen 
will  show  the  oval  bacilli. 

Recent  investigations  in  India  have  definitely  determined  the  fact 
that  the  flea  (Pulex  cheopis)  is  the  intermediary  in  the  transmission  of 
plague  from  rat  to  rat  and  from  rat  to  man.  In  pneumonic  plague  the 
infective  nature  is  very  great  and  appears  to  be  by  the  respiratory 
atrium.  This  was  the  terrifying  type  of  plague  in  the  black  death  of 
the  fourteenth  centurv. 


FIG.  32. — Pest  bacillus  involution  forms  produced  by  growing  on  3%  salt  agar. 
(Kolle  and  Wassermann.} 

For  diagnosis  make  smears  and  cultures  from  material  drawn  from 
a  bubo  by  a  syringe.  (At  a  later  stage,  when  softening  begins,  there 
may  not  be  any  bacilli  present.)  Also,  if  pneumonic  plague,  from  the 
sputum.  Blood  cultures  and  even  blood  smears  may  be  employed  in 
septicaemia  plague.  Formol  fuchsin  makes  a  satisfactory  stain. 
Always  inoculate  a  guinea-pig  with  the  material  either  by  rubbing  it  in 
with  a  glass  spatula  on  the  shaven  skin  or  by  subcutaneous  injection. 
For  prophylaxis  the  most  important  method  is  that  of  Haffkine. 
Stalactite  bouillon  cultures  of  plague  are  grown  for  five  to  six  weeks. 
These  are  killed  by  a  temperature  of  65°  C.  for  one  hour.  Lysol  (J%)  is 
added  to  the  preparation  and  from  0.5  to  4  c.c.  injected,  according  to 


TYPHOID COLON  GROUPS.  85 

tin-  age  and  size  of  the  individual  treated.  Susceptibility  is  reduced 
about  one-fourth,  and  of  those  attacked  after  previous  vaccination,  the 
mortality  is  only  about  one-fourth  of  what  it  is  among  the  noninocu- 
lated.  'Strong  prepares  a  prophylactic  vaccine  from  living  plague 
cultures  rendered  avirulent.  Yersin's  serum,  made  by  injecting  horses 
with  dead  plague  cultures  and  afterward  with  living  ones,  is  of  value 
prophylactically  and  has  possibly  considerable  curative  power. 

The  Eberth,  Gartner  and  Escherich  Groups. — From  a  stand- 
point of  cultures  in  litmus  milk  and  sugar  bouillon  we  can  divide  the 
organisms  related  to  typhoid  at  one  extreme  and  the  colon  at  the  other 
into  three  groups. 

1.  The   Eberth   or   typhoid   group.     There   are  three  important 
pathogens  in  this  group:  the  B.  typhosus,  the  B.  dysenteriae,  and  the 
B.  fecalis  alkaligenes.     The  color  of  litmus  milk  is  practically  unal- 
tered, and  there  is  no  gas  production  in  either  glucose  or  lactose  bouillon. 
No   coagulation   of   milk.     No   reduction   of  neutral   red.     The   B. 
typhosus  and  the  B.  fecalis  alkaligenes  are  actively  motile,  while  the  B. 
dysenteriae  is  nonmotile  or  practically  so. 

During  the  first  24  to  48  hours  there  is  a  moderate  acid  production 
by  typhoid,  so  that  the  milk  culture  is  less  blue,  while  with  the  B. 
fecalis  alkaligenes  the  alkalinity  is  intensified  from  the  start  so  that 
the  blue  color  is  deepened. 

2.  The  Gartner  or  hog  cholera  group.     Besides  organisms  im- 
portant for  animals  and  probably  at  times  for  man,  such  as  B.  cholerae 
suum  and  B.  psittacosis  and  B.  icteroides  (interesting  historically  as 
having  been  reported  as  the  cause  of  yellow  fever  by  Sanarelli),  we  have 
two  pathogens:    (i)  B.   enteritidis  (Gartner's   bacillus)   and    (2)    B. 
paratyphoid  B.     These  organisms  cannot  be  separated  culturally,  but 
only  by  immunity  reactions.     They  do  not  tarn  litmus  milk  pink. 
They  produce  gas  in  glucose  bouillon,  but  not  in  lactose.     They  very 
powerfully  reduce  neutral  red  with  the  production  of  a  yellowish 
fluorescence.     They  do  not  coagulate  milk.     There  is  a   transient 
acidity  in  the  litmus  milk,  but  becoming  shortly  afterward  alkaline, 
the  lilac-blue  color  is  intensified.     Both  organisms  are  motile. 

3.  The  Escherich  or  colon  group.     These  turn  litmus  milk  pink, 
coagulate  milk,   reduce  neutral  red    and    show  varying  degrees  of 


86 


STUDY    AND    IDENTIFICATION    OF    BACTERIA. 


motility.  The  three  groups  of  organisms  just  described  are  non- 
liquefiers  of  gelatin.  Two  intestinal  organisms,  the  B.  cloacae  and  the 
Proteus  vulgaris,  differ  in  liquefying  gelatin. 

Bacillus  Typhosus  (Eberth,  1880;  Gaffky,  1884).— This  organism 
may  be  isolated  from  the  stools,  urine  and  the  blood  of  typhoid 
patients.  At  postmortem  it  can  be  best  isolated  from  the  spleen,  but  is 
also  present  in  Peyer's  patches  which  have  not  ulcerated.  When 
ulceration  has  occurred  contamination  with  B.  coli  is  almost  sure. 
Cultures  may  also  be  obtained  from  the  liver.  In  sections  made  from 
spleen  the  Gram  negative  bacilli  are  apt  to  be  decolorized.  Thionin, 


FIG.  33. — Seventy-two  hour  old  culture  of  typhoid  bacillus  on  gelatine. 
(Kolle  and  Wassermann.} 

then  blotting  and  clearing  in  oil  or  xylol,  shows  the  clumps  of  bacilli 
lying  between  the  cells. 

Formerly  it  was  supposed  that  by  the  differences  in  the  thickness  of 
the  film  of  a  colony  or  by  its  varying  shades  of  grayish-blue,  we  pos- 
sessed data  of  importance  in  differentiating  typhoid  from  related 
organisms.  Growth  on  potato  was  also  considered  as  affording  in- 
formation. At  present,  the  biochemical  reactions  give  us  information 
assisting  in  differentiation,  and  the  agglutination  or  bacteriolytic 
phenomena,  the  final  diagnosis.  The  various  plating  media  are  con- 
sidered under  the  studv  of  feces. 


TYPHOID.  87 

Not  only  do  we  find  hyperplasia  of  the  endothelial  cells  in  the 
lymphoid  tissue  of  Peyer's  patches  and  the  mesenteric  glands  and  the 
spleen,  with  subsequent  necroses,  but  focal  necroses  of  the  same 
character  are  found  in  the  liver. 

A  striking  feature  of  the  pathology  of  typhoid  fever  is  the  long- 
continued  persistence  of  the  organisms  in  the  gall-bladder  and  else- 
where. It  is  beginning  to  be  believed  that  a  previous  typhoid  infection, 
possibly  so  mild  as  to  have  passed  unnoticed,  is  at  the  basis  of  gall- 
bladder infections  and  resulting  gall-stones.  Various  bone  infections, 
especially  osteomyelitis,  have  shown  the  typhoid  bacilli  in  pure  culture. 
Formerly  it  was  supposed  that  the  typhoid  bacillus  brought  about  its 
lesions  by  a  local  infection  centered  in  the  ileum.  The  present  view 
is  that  typhoid  bacilli  effect  an  entrance  into  the  blood  stream  through 
some  lymphoid  channel,  as  by  tonsil  or  other  alimentary  lymphoid 
structure.  It  multiplies  in  the  blood  during  the  period  of  incubation  and 
it  is  only  when  bactericidal  properties  are  developed  in  the  blood  that 
we  have  destruction  of  the  bacilli  and  liberation  of  the  intracellular 
toxins  which  lead  to  the  development  of  symptoms.  That  this  is 
probable  is  shown  by  the  fact  that  typhoid  bacilli  can  be  isolated  from 
the  blood  during  the  period  of  incubation.  It  is  a  practical  point  that 
the  time  to  isolate  the  bacteria  from  the  blood  is  in  the  first  days  of  the 
attack.  The  diagnosis  by  agglutination  is  only  expected  after  the 
seventh  to  tenth  day.  Agglutination  may  not  appear  until  during  con- 
valescence, and  in  about  5%  of  the  cases  it  is  absent.  It,  as  a  rule, 
disappears  within  a  year.  Very  little  success  has  been  obtained  with 
curative  sera.  Chantamesse,  by  treating  horses  with  a  filtrate  from 
cultures  of  typhoid  bacilli  on  splenic  pulp  and  human  defibrinated 
blood,  claimed  to  have  obtained  a  curative  serum  possessing  antitoxic 
power.  Wright's  method  of  prophylactic  inoculation  is  now  being 
employed  in  the  British  army  with  apparent  success.  In  this,  24-  to  48- 
hour-old  cultures  are  killed  at  53°  C. ;  1/4%  of  lysol  is  then  added.  An 
injection  of  500  million  bacteria  is  made  at  the  first  inoculation,  and 
ten  days  later  an  injection  of  one  billion.  The  British  prefer  to  inject 
subcutaneously  in  the  infraclavicular  region  and  at  the  insertion  of  the 
deltoid.  The  Germans  consider  three  injections  as  conferring  greater 
immunity. 


88  STUDY   AND    IDENTIFICATION    OF    BACTERIA. 

A  very  important  discovery  is  that  certain  persons,  who  may  have 
had  only  a  slight  febrile  attack,  may  eliminate  typhoid  bacilli  for  years 
in  their  feces  (typhoid  carriers).  The  bacilli  are  also  eliminated  for 
considerable  periods  in  the  urine.  Distinction  is  now  being  made 
between  acute  carriers  (convalescents)  and  chronic  carriers.  Or- 
dinarily, it  is  very  difficult  to  isolate  typhoid  bacilli  from  the  stools. 
For  laboratory  diagnosis,  blood  cultures  during  the  first  week  and 

„•  -^       v1 

-   "    \ 


HE  •'•  S  ?  V^"-  -  O   S 


^C/:;;..? 

FIG.  34. — Bacillus  of  typhoid  fever,  stained  by  Loffler's  method  to  show 
flagella.     (X  1000.)     (Williams.) 


agglutination  tests  during  the    second  week    and  onward   are    the 
practical  methods.     B.  typhosus  appears  in  the  blood  in  relapses. 

Paratyphoid  Bacilli  (Achard  and  Bensaude,  1896;  Schottmiiller, 
1901). — Cases  resembling  mild  attacks  of  typhoid  occasionally  show 
agglutination  for  paratyphoid  bacilli.  These  organisms  have  also 
been  isolated  from  the  blood,  as  with  typhoid.  Two  types  have  been 
recognized:  the  paratyphoid  A  and  the  paratyphoid  B.  The  latter 
occurs  in  80%  of  such  cases.  Culturally,  paratyphoid  B  cannot  be 


PARATYPHOID  AND    DYSENTERY.  89 

r]>a rated  from  Gartner's  bacillus.  In  paratyphoid  A  there  is  less  gas 
produced  in  glucose  bouillon  than  with  paratyphoid  B,  and  the  primary 
acidity  of  litmus  milk  is  not  succeeded  by  a  subsequent  alkalinity.  It 
does  not  seem  practical  to  draw  a  fine  distinction  between  these  two 
strains.  Bacillus  Enteritidis  (Gartner,  1888).  This  organism  has 
been  frequently  isolated  from  cases  of  gastroenteritis  from  ingestion 
of  infected  meat.  This  organism  is  very  pathogenic  for  laboratory 
animals,  producing  a  hemorrhagic  enteritis  and  at  times  a  septicaemia. 
Where  meat  has  been  contaminated  with  Gartner's  bacillus  toxins  may 
have  been  produced,  and  symptoms  of  poisoning  with  acute  gastro- 
enteritis would  occur  shortly  after  ingestion.  It  is  interesting  to  note 
that  this  toxin  is  not  destroyed  by  the  boiling  temperature,  thus  differ- 
ing from  the  toxin  of  the  other  important  meat  poisoning  (botulism) 
bacillus — B.  botulinus — which  is  rendered  innocuous  by  a  temperature 
of  65°  or  70°  C.  If  there  is  only  a  little  toxin  introduced  with  the  con- 
taminated meat,  the  symptoms  will  be  delayed  one  or  two  days.  Such 
organisms  have  been  isolated  in  pure  culture  from  cases  with  high 
fever,  marked  intestinal  derangement,  with  considerable  blood  in  the 
rather  fluid  stools.  In  two  cases  studied  the  disease  was  at  first 
diagnosed  as  a  severe  typhoid  infection.  Klein  thinks  the  organism  of 
Danysz's  virus  (to  kill  rats  during  plague  epidemics)  may  be  identical 
with  B.  enteritidis. 

Proteus  Vulgaris. — This  organism  is  often  encountered  in  plates 
made  from  feces.  It  is  common  in  decaying  meat  or  cheese,  and  cases 
of  even  fatal  poisoning  with  marked  gastrointestinal  symptoms  and 
cardiac  failure  have  been  reported.  At  times  it  is  the  cause  of  cystitis. 
The  colonies  on  agar  are  moist  and  unevenly  spreading  (amoeboid). 
The  bacilli  are  very  motile  and,  as  a  rule,  Gram  negative.  It  digests 
blood  serum  and  is  a  rapid  liquefier  of  gelatin.  The  cultures  generally 
have  a  putrefactive  odor.  In  infective  jaundice  (Weil's  disease)  this 
organism  has  been  reported  as  the  cause. 

Bacillus  Dysenteriae  (Shiga,  1898). — Dysentery  bacilli  produce 
a  coagulation  necrosis  of  the  mucous  membrane  of  the  large  intestine 
and  occasionally  of  the  lower  part  of  the  ileum.  Polymorphonuclears 
are  contained  in  the  fibrin  exudate.  It  was  formerly  thought  that  these 
lesions  were  of  local  origin,  but  the  present  view  is  that  toxins  are 


QO  STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

produced  which,  being  absorbed,  are  eliminated  by  the  large  intestine 
with  resulting  necrosis.  Flexner,  by  injecting  rabbits  intravenously 
with  a  toxic  autolysate,  produced  characteristic  intestinal  lesions. 
There  are  two  main  types  of  dysentery  bacilli : 

1.  Those   producing   acid   in   mannite   media — the    acid   strains 
(Flexner-Strong  types). 

2.  Those  not  developing  acid   in  mannite  (Shiga-Kruse  types). 
Ohno  finds  that  fermentative  reactions  do  not  correspond  to  immunity 
ones.     Thus  an  acid  strain  used  to  immunize  a  horse  may  produce  a 
serum  more  specific  for  a  nonacid  strain.     The  Shiga  type  is  very 
toxic  in  cultures,  while  the  Flexner  type  does  not  seem  to  possess  a 
soluble  toxin.     At  the  Lister  Institute  injections  of  a  soluble  toxin 
produced  a  serum  of  marked  antitoxic  power.     Such  a  dysentery 
serum,  which  is  probably  both  antitoxic  and  antimicrobic,  is  of  curative 
value.     Shiga  immunized  horses  with  polyvalent  cultures  and  obtained 
a  polyvalent  serum  which  has  reduced  the  death-rate  about  one-third. 

The  dysentery  bacillus  is  present  in  the  mucous  stools  during  the 
first  five  or  six  days  of  the  disease.  By  the  tenth  day  it  has  probably 
disappeared.  In  all  cultural  respects  the  dysentery  bacillus  resembles 
the  typhoid,  and  the  only  practical  method  of  distinguishing  these  two 
organisms,  other  than  by  agglutination  reactions,  is  by  the  nonmotility 
or  exceedingly  slight  motility  of  the  dysentery  bacillus.  The  dysentery 
bacilli  do  not  form  those  threads  or  whip-like  filaments  so  characteristic 
of  typhoid  cultures.  The  dysentery  bacillus  is  not  found  in  the  blood 
and  hence  is  not  eliminated  in  the  urine.  It  is  found  in  mesenteric 
glands.  In  dysentery  patients  agglutination  phenomena  do  not  show 
themselves  until  about  the  twelfth  day  from  the  onset.  Hence,  this 
procedure  is  of  no  particular  value  in  diagnosis.  It  is  of  value,  how- 
ever, to  identify  an  organism  isolated  from  the  stools  at  the  commence- 
ment of  the  attack,  using  serum  from  an  immunized  animal  or  a 
human  convalescent  for  the  agglutination  test. 

Park  and  Collins,  in  1904,  separated  various  strains  of  dysentery 
bacilli  into  three  groups: 

i.  Original  Shiga  strain.     No  indol.     No  fermentation  of 
mannite,  maltose  or  saccharose. 


THE  ROLE  OF  THE  COLON  BACILLUS.  91 

2.  A  type  fermenting  mannite,  but  not  maltose  or  saccharose. 
No  indol. 

3.  An  indol  producer  which  actively  ferments  mannite  and 
maltose, 

B.  COLI,  B.  LACTIS  AEROGENES,  B.  CLOACAE. 

While  the  colon  bacillus  chiefly  inhabits  the  large  intestine,  the  B. 
lactis  aerogenes  is  to  be  found  in  the  upper  part  of  the  small  intestine. 
While  they  may  be  separated  on  the  ground  of  motility,  yet  it  is  by  the 
greater  fermentative  activity  of  the  B.  lactis  aerogenes  that  they  are 
best  separated.  Some  consider  them  as  only  representing  different 
strains  of  the  same  organism.  Some  consider  that  the  B.  coli  produces 
a  bactericidal  substance  which  inhibits  the  growth  of,  or  destroys 
pathogenic  bacteria  which  may  have  passed  the  destructive  influences 
of  the  gastric  fluid;  others  that  this  effect  is  due  to  their  free  growth 
and  the  development  of  phenol  and  various  putrefactive  substances. 
The  probable  importance  of  the  colon  bacillus  in  protecting  the  organ- 
ism is  shown  by  the  fact  that  where  numerous  colonies  of  pathogenic 
organisms  may  be  cultivated  from  feces  we  may  find  a  diminution  in 
number  or  absence  of  the  colon  bacillus.  This  condition  may  be 
observed  in  infections  with  the  organisms  of  dysentery,  cholera,  typhoid 
and  paratyphoid.  While  its  normal  function  is  probably  protective, 
yet  the  B.  coli  is  an  important  pathogenic  agent,  it  being  frequently 
the  organism  isolated  from  purulent  conditions  within  the  abdominal 
cavity,  especially  in  appendicitis  and  lesions  about  the  bile  ducts. 
It  is  particularly  prone  to  cause  lesions  of  the  bladder  and  pelvis  of 
the  kidn.ey.  In  the  treatment  of  colon  cystitis  by  vaccines  of  dead 
colon  bacilli,  the  most  brilliant  results  in  opsonic  therapy  have  been 
obtained. 

Sir  A.  Wright  thinks  that  certain  cases  of  mucous  colitis  may  be 
due  to  colon  infection  and  that  vaccination  may  cure  them.  The 
colon  bacillus  is  fully  considered  under  the  bacteriology  of  water. 

B.  cloacae  was  isolated  first  from  sewage  by  Jordan.  It  is  a  rapid 
liquefier  of  gelatin,  and  in  its  reaction^  with  sugars  and  litmus  milk 
resembles  the  colon  bacillus. 


92  STUDY  AND  IDENTIFICATION  OF  BACTERIA. 

CHROMOGENIC  BACILLI. 

These  are  identified  by  the  color  of  their  colonies  on  agar.  The 
B.  pyocyaneus  is  the  most  important  one  of  them  in  medicine,  but 
the  B.  prodigiosus  is  also  of  interest  medically.  A  violet  chromogen, 
the  B.  violaceus,  which  is  motile  and  liquefies  gelatin,  has  been  de- 
scribed under  many  names.  It  has  been  found  in  water. 

An  orange-yellow  chromogen,  the  B.  fulvus,  is  nonmotile  and 
varies  as  to  its  liquefaction  of  gelatin. 

B.  pyocyaneus  (Gessard,  1882). — This  organism  is  frequently 
termed  the  bacillus  of  green  or  blue  pus.  It  is  a  small  (2.5  x  .$/*) 


FIG.  35. — Bacillus  pyocyaneus.     (Kolle  and  Wassermann.) 

motile  Gram  negative  bacillus.  It  grows  readily  at  room  or  incubator 
temperature.  It  liquefies  gelatin  rapidly.  The  green  color  diffuses 
through  the  agar  or  gelatin  on  which  it  grows,  so  that  we  not  only 
have  the  green-colored  colony,  but  the  medium  as  well  is  colored. 
Upon  potato  the  colonies  are  more  of  a  dirty  brown.  It  is  widely 
distributed  in  water  and  air,  and  is  frequently  isolated  from  faeces. 
The  B.  fluorescens  liquefaciens  of  water  seems  to  be  simply  a  strain 
of  B.  pyocyaneus.  The  B.  pyocyaneus  is  frequently  associated  with 
other  pus  organisms  in  abdominal  abscesses.  In  addition  to  having 
an  endo toxin,  it  produces  a  soluble  toxin  similar  to  diphtheria  toxin. 


CHROMOGfiNIC    BACILLI.  93 

This  toxin  differs  from  those  of  diphtheria  and  tetanus  in  that  it  can 
stand  a  temperature  of  100°  C.,  while  those  of  diphtheria  and  tetanus 
are  destroyed  at  about  65°  C.  The  fact  that  the  union  between  toxin 
and  antitoxin  is  only  of  a  binding,  neutralizing  nature  is  best  shown 
by  taking  a  mixture  of  pyocyaneus  toxin  and  antitoxin,  which  is  innocu- 
ous and  heating  it.  This  destroys  the  antitoxin,  but  does  not  injure  the 
toxin.  We  now  find  that  the  original  toxicity  has  returned.  The 
antitoxins  of  diphtheria  and  tetanus  are  more  stable  than  the  corre- 
sponding toxins;  hence,  this  experiment  would  be  impossible  with 
them,  as  upon  heating  we  should  fir  >t  destroy  the  toxin. 

B.  prodigiosus.  This  is  a  very  small  coccobacillus  which  shows 
motility  in  young  bouillon  cultures.  It  is  Gram  negative.  The 
colonies  on  agar  or  other  solid  media  show  a  rich  red  color.  The 
pigment  only  develops  at  room  temperature;  it  is  absent  in  cultures 
taken  out  of  the  incubator.  The  B.  prodigiosus  is  frequently  found 
on  food  stuffs,  especially  bread,  where  it  may  simulate  blood.  It 
liquefies  gelatin  rapidly  and  gives  a  diffuse  turbidity  to  bouillon.  It  is 
probable  that  B.  indicus  and  B.  kilensis  are  strains  of  B.  prodigiosus. 

Coley's  fluid,  which  has  been  used  in  cases  of  inoperable  sarcoma 
and  other  malignant  growths,  is  a  culture  prepared  by  growing  very 
virulent  streptococci  in  bouillon  for  ten  days.  This  streptococcus 
culture  is  now  inoculated  with  B.  prodigiosus,  and  after  another  ten 
days  the  mixed  culture  is  killed  by  heat  at  60°  C.  and  the  sterile 
product  injected.  Coley  injected  about  one-twentieth  of  a  c.c.  of 
this  vaccine. 


CHAPTER  IX. 

STUDY  AND  IDENTIFICATION  OF  BACTERIA.     SPIRILLA. 
KEY  AND  NOTES. 

Key  to  recognition  of  gelatin-liquefying,  motile  and  Gram  negative 
spiral  or  comma-shaped  organisms. 

A.  Do  not  give  the  nitroso-indol  reaction  with  sulphuric  acid  alone  in  24 

hours. 

1.  Produce  an  abundant  moist  cream-colored  growth  on  potato  at  room 
temperature. 

a.  Finkler  and  Prior's  spirillum.  Liquefaction  of  gelatin  very  rapid. 
No  air-bubble  appearance  at  top  of  liquefied  area.  Cultures  have 
foul  odor.  Milk  coagulated.  Thicker  spirillum  than  cholera. 
Isolated  from  cholera  nostras. 

2.  Scanty  growth  or  none  at  all  on  potato  at  room  temperature.     Only 

a   moderate   yellowish  growth  when   incubated   about  incubator 
temperature. 

a.  Spirillum  tyrogenum.  Does  not  liquefy  gelatin  so  rapidly  as 
Finkler  Prior.  Thinner  and  smaller  spirillum  than  cholera. 

B.  Give  the  nitroso-indol  reaction  with  sulphuric  acid  within  24  hours. 

1.  Very  pathogenic  for  pigeons. 

a.  Spirillum  metschnikovi.  Liquefies  gelatin  about  twice  as  rapidly 
as  cholera.  Gives  bubble  appearance  at  top  of  stab. 

2.  Scarcely  pathogenic  for  pigeons. 

a.  Spirillum   cholerse   asiaticas.     Milk   not  coagulated. 

Nonmotile,  nonliquefying  gelatin  and  Gram  positive  spirilla  have  also  been 
described.  There  is  also  a  large  group  of  phosphorescent  spirilla. 

Spirillum  Choleras  Asiaticae  (Koch,  1884). — Typically,  the 
morphology  of  this  organism  is  that  of  the  comma  (Comma  bacillus 
of  Koch).  It  also  frequently  shows  S  shapes,  and  often  appears  in 
long  threads  showing  turns.  When  freshly  isolated  from  cholera 
material  they,  as  a  rule,  show  a  fairly  typical  morphology,  but  after 
subcultures  in  the  laboratory  variations  are  common,  so  that  red  forms 
and  round  involution  shapes  give  a  picture  altogether  at  variance 
with  the  comma  shape.  Even  in  recent  cultures  of  undoubted  cholera 
we  may  have  different  types,  as  coccoid  forms  and  slender  rods. 

The  cholera  spirillum  is  very  motile  and  liquefies  gelatin  fairly 

94 


CHOLERA. 


95 


rapidly,  although  more  slowly  than  any  of  the  spirilla  mentioned  in 
the  key.  The  colony  on  gelatin  was  formerly  considered  charac- 
teristic, but  like  most  cultural  characteristics,  it  is  now  considered  as 


FIG.  36. — Cholera  spirilla.     (Kolle  and  Wassermann.} 

being  only  of  confirmatory  value;  it  is  not  specific.  These  colonies 
show  in  24  hours  as  small  granular  white  spots  which  have  a  spinose 
periphery.  An  encircling  ring  of  liquefaction  now  makes  its  appear- 


FIG.  37. — Involution  forms  of  the  spirillum  of  cholera.  (Van  Ermengem.) 

ance  and  the  highly  refractile  (as  if  fragments  of  sparkling  glass) 
colony  can  be  separated  into  a  granular  center,  a  striated  periphery 
and  a  clear  external  ring  of  liquefaction. 


96 


STUDY    AND    IDENTIFICATION    OF    BACTERIA. 


On  gelatin  stabs  the  liquefaction  produces  a  turnip -like  hollow 
at  the  top  of  the  puncture — the  air  bubble  appearance.  It  gives  the 
nitroso-indol  reaction  with  sulphuric  acid  alone  (cholera  red).  Kraus 
attaches  importance  to  the  fact  that  cholera  does  not  produce  a 
haemolytic  ring  on  blood  agar  as  do  the  pseudo- 
cholera  spirilla ;  a  difficulty  is  that  many  pseudo- 
spirilla  do  not  haemolize.  It  grows  very  rapidly  on 
peptone  solution  and  this  is  the  medium  for  the 
enrichment  test  to  be  later  described.  On  this  it 
may  form  a  pellicle.  On  agar  the  colony  is  more 
opalescent  than  the  typhoid.  It  does  not  grow  on 
potato  except  at  incubator  temperature.  It  does 
not  coagulate  or  turn  acid  litmus  milk.  The  spirilla 
are  found  in  myriads  in  the  rice-water  discharges, 
these  white  flakes  being  desquamated  epithelial  cells. 
They  penetrate  the  crypts  of  Lieberkuhn,  but  rarely 
extend  to  the  submucosa.  The  symptoms  are  due 
to  an  endotoxin. 

Cholera  may  be  transmitted  from  water  supplies, 
when  the  outbreak  is  apt  to  be  widespread  and  in 
great  numbers  from  the  start.  Also  by  indirect 
contagion,  as  by  flies  or  on  lettuce,  etc.  A  very 
important  point  is  that  we  have  well  persons  whose 
faeces  contain  virulent  cholera  spirilla  (cholera  carriers).  To  identify 
such  spirilla  immunity  reactions  are  necessary: 

1.  Injected  intraperitoneally  into  guinea-pigs,  it  produces  a 
peritonitis   and   subnormal   temperature.     This  reaction 
exists  for  spirilla  other  than  the  true  cholera  spirillum. 

2.  Intramuscular  injections  into  pigeons  are  only  slightly 
pathogenic,  if  at  all. 

3.  The  agglutination  test  is  the  most  practical.     In    this  we 
use  serum  from  an  immunized  animal,  in  dilution  of  from 
50  to  1000.     Serum  of  cholera  convalescents  may  show 
agglutination  as  early  as  the  tenth  day;  it  is  usually  best 
shown  about  the  third  week.     Dunbar's  quick  method  is 
very    practical.     Make    two   hanging- drop   preparations, 


FIG.  38.— Spiril- 
lum of  cholera, 
stub  -  culture  in 
gelatin,  two  days 
old.  (Frankel  and 
Pfeiffer.} 


CHOLERA.  97 

using  mucus  from  the  stool  as  the  bacillary  emulsion. 
To  one  add  an  equal  amount  of  a  1:50  normal  serum; 
to  the  other  a  i :  500  dilution  of  immune  serum.  Cholera 
spirilla  remain  motile  in  the  control,  but  lose  motility  and 
become  agglutinated  in  the  preparation  with  the  immune 
serum. 

4.  Pfeiffer's  phenomenon.  If  cholera  spirilla  are  introduced 
into  the  peritoneal  cavity  of  immunized  guinea-pigs  (or  if 
together  with  a  i :  1000  dilution  of  immune  serum  the 
mixture  is  injected  intraperitoneally  into  normal  guinea- 
pigs)  and  at  periods  of  ten  to  sixty  minutes  after  injection, 
material  is  removed  by  a  pipette  from  the  peritoneal 
cavity,  the  spirilla  have  lost  motility,  have  become  granular 
and  degenerated.  Pseudospirilla  are  unchanged.  This 
reaction  may  be  carried  on  in  a  pipette,  using  fresh  serum. 
Antisera  for  the  treatment  of  cholera  have  not  proved 
successful.  Prophylactically,  there  are  two  prominent 
methods:  (T)  That  of  Haffkine,  where  live  cholera  spirilla 
are  injected  subcutaneously;  and  (2)  Strong's  cholera 
autolysate.  In  this  cholera  cultures  are  killed  at  60°  C. 
The  killed  culture  is  then  allowed  to  digest  itself  in  the 
incubator  at  37°  C.  for  three  or  four  days  (peptonization). 
The  preparation  is  then  filtered  and  from  2  to  5  c.c.  of  the 
filtrate  is  injected. 
For  diagnosis  take  a  fleck  of  mucus,  make  a  straight  smear  and  fix; 

stain   with   a  i :  10  carbol  fuchsin.     The  comma  shaped  organisms 

appear  as  fish  swimming  in  a  stream. 

2.  Inoculate    a    tube    of    peptone    solution.     The    cholera 
spirilla  grow  so  rapidly,  and  being  strong  aerobes,  they 
grow  on  the  surface  of  the  fluid  so  that  by  taking  a  loopful 
from  the  surface,  we  may  in  eight  hours  obtain  a  pure 
culture.     Inoculate  a  second  tube  from  this  first  and, 
if  necessary,  a  third  (enrichment  method). 

3.  Test  for  cholera  red  reaction.     (Simply  adding  from  three 
to  five  drops  of  concentrated  sulphuric  acid  in  the  first 
or  second  peptone  culture  after  18  to  24  hours'  growth.) 


STUDY    AND    IDENTIFICATION    OF    BACTERIA. 

At  times  we  only  get  the  cholera  red  when  we  have  a  pure 
culture  of  cholera. 

4.  Smear  a  fleck  of  mucus  or,  better,  the  surface  growth  on 
peptone,  on  a  dry  agar  surface  in  a  Petri  dish.     From 
colonies  developing,  make  agglutination  and,  if  desired, 
cultural  tests.     It  is  by  immune  reactions  that  we  identify 
cholera  spirilla.     The  surface  moisture  of  plates  is  best 

dried  by  the  filter-paper  top. 

5.  For  methods  with  water,  see  Water  Analysis. 


CHAPTER  X. 


STUDY  AND  IDENTIFICATION  OF  MOULDS. 

CLASSIFICATION  OF  THE  FUNGI. 


Order  Suborder 

Phycomycetes      Zygomycetes 


Gymnoascus 


Ascomvcetes 


Carpoascus 


Hyphomycetes 


Family                 Genus 

Species 

Mucor 

'  M.  corymbifer 
M.  mucedo 

f  S.  cerevisiae 

Saccharomyces  J 

S.  anginae 

[  S.  blanchardi 

Saccharo- 
mycetes 

Endomyces 

E.  albicans 

Cryptococcus 

f  C.  gilchristi 
C.  hominis 

T.  sabouraudi 

T.  tonsurans 

'  Trichophyton 

T.  violaceum 

T.  mentagro- 

Gymno- 

phytes 

asci 

T.  cruris 

Microsporum 

M.  audouini 

Achorion 

A.  schoenleini 

Penicillium 

P.  crustaceum 

rA.  fumigatus 

Perisporia- 

Aspergillus 

A.  concentricus 

ceae 

'> 

A.  pictor 

A.  niger 

Discomyces         < 

D.  bovis 
D.    madurae 

Madurella 

M.  raycetomi 

Malassezia 

M.  furfur 

Microsporoides 

M.  minutissimus 

Trichosporum 

T.  giganteuin 

THE  FUNGI. 

The  Thallophyta  are  plants  in  which  there  is  no  differentiation 
between  root  and  stem. 

The  classes  of  Thallophyta  which  are  of  interest  medically  are  (i) 
the  Algae  and  (2)  the  Fungi. 

99 


100  STUDY   AND    IDENTIFICATION    OF    MOULDS. 

Some  include  Lichenes  as  a  separate  class.  These  are  really 
symbiotic  organisms — Fungi  parasitic  on  Algae. 

The  Algae  contain  chlorophyll,  with  the  exception  of  Cyanophy- 
ceae.  To  the  order  Cyanophyceae  it  is  considered  that  the  family  of 
Bacteria  belong. 

The  fungi  do  not  possess  chlorophyll.  They  are  in  their  simplest 
forms  ramifying  filaments  called  hyphae.  The  vegetative  hyphae 
which  intertwine  in  tangled  threads,  as  a  support,  are  termed  the 
mycelium,  while  those  which  project  upward  are  called  the  aerial 
hyphae  and  are  the  ones  wrhich  bear  the  conidia  or  spores. 

The  orders  of  the  class  fungi  which  are  of  interest  medically  are:  (i) 
the  Phycomycetes;  (2)  the  Ascomycetes;  (3)  the  Hyphomycetes. 

Phycomycetes. — These  produce  a  copious  mycelium,  bear  conidia, 
and  reproduce  in  the  case  of  the  suborder  Oomycetes  by  heterogamy. 
(Dissimilar  sexual  cells — a  smaller  male,  antheridium,  and  a  larger 
female,  oogonium.  By  fertilization  by  antherozoids  from  the  anther- 
idium penetrating  the  oosphere  we  have  oospores.) 

The  suborder  Zygomycetes  reproduces  either  asexually  or  by 
isogamy  (two  similar  sexual  cells  conjugate  and  form  on  fusion  a 
Zygospore.)  Of  these  wre  have  two  species  of  the  genus  Mucor:  (i) 
Mucor  mucedo  and  (2)  Mucor  corymbifer.  These  moulds  develop 
especially  in  external  cavities.  Pulmonary  and  generalized  infections 
have  also  been  reported.  The  pathogenic  species  have  smaller  spores 
and  grow  best  at  37°  C.  The  thick,  coarse,  cotton-like  mould  seen  on 
horse  manure  is  a  Mucor.  The  sporangium,  the  organ  of  fructification, 
contains  the  spores  within  its  interior.  The  M.  mucedo  has  thick 
silver-gray  mycelium,  with  large  sporangia,  150/4  in  diameter,  contain- 
ing oval  spores,  5  x  9/4.  The  M.  corymbifer,  which  has  been  re- 
ported from  a  generalized  infection,  considered  as  typhoid,  shows  a 
snow-white  mycelium.  The  sporangia  are  20  to  40/4  and  the  spore 
about  3/z  in  diameter. 

Ascomycetes. — In  this  order  are  included  many  of  the  parasitic 
moulds.  The  most  distinctive  characteristic  is  the  formation  of 
ascospores  in  an  ascus  (little  sac).  It  is  an  elongated  sporangium  in 
which  a  definite  number  of  spores,  usually  eight,  is  formed.  The 
ascus  usually  ruptures  at  its  tip.  Other  members  of  the  order  are 


TEASTS.  10 I 

formed  from  hyprue  by  the  separation  of  cells  in  succession  from  the 
free  ends. 

The  order  is  divided  into  those  with  naked  asci  (Gymnoascus)  and 
those  having  a  perithecium  or  investing  layer  (Carpoascus). 

Belonging  to  the  suborder  Gymnoascus  we  have  (i)  the  family  of 
Saccharomycetes,  which  reproduce  by  budding  and  in  which  the  asci 
are  without  any  semblance  of  a  sheath,  and  (2)  a  family  in  which  there 
is  an  indication  of  the  formation  of  a  perithecium — this  may  be  termed 
the  Gvmnoasci  familv. 


FIG.  39  — Yeast  cells.     Saccharomyces  cerevisiae.     (Coplin.) 

Saccharomycetes. — There   are   three   genera:  Saccharomyces,    En- 
do  myces  and  Cryptococcus. 
Saceharomyces. — These  have  ascospores. 

• — Sr- cerevisiae. — This  is  the  ordinary  yeast  fungus.     Used  at 

times  as  an  antiseptic. 
S.  anginae. — Found  in  a  case  of  angina. 
S.  blanchardi. — Found  in  a   jelly-like  tumor  mass  of    the 
abdomen.     The   budding   cells   varied   from  2  to 
2o/£.     Probably  identical  with  S.  tumefaciens. 
Endomyces. — Forms  spores  in  the  interior  of  filaments. 

E.  albicans. — The  organism  of  thrush.     It  produces  a  false 
membrane,  especially  on  buccal  surfaces.     Grows 


102  STUDY    AND    IDENTIFICATION    OF    MOULDS. 

only  in  acid  media.     Hence  propriety  of  alkaline 
treatment. 

Cryptococcus. — Reproduces  by  budding,  but  ascospore  formation  not 
observed.  Not  a  well-recognized  genus.  The  diseases 
caused  by  it  are  termed  blastomycoses. 

C.  Gilchristi. — The  cells  are  about  i6/z  in  diameter  and  have 
a  thick  membrane.  They  reproduce  by  budding. 
May  invade  internal  organs.  It  is  cultivable.  A 
mould,  somewhat  similar,  is  the  Coccidioides  im- 


FIG.  40. — Thrush  fungus.     (Kolle  and  Wassermann.) 

mitis  of  Ophuls.  This  has  a  mycelial  growth. 
The  infection  frequently  becomes  generalized. 
The  small  bodies,  about  3//,  in  the  Molluscum  con- 
tagiosum  cells  are  thought  by  some  to  be  yeasts. 
They  are  more  probably  artefacts.  Plimmer's 
bodies  in  cancer  cells  belong  in  this  group.  They 
also  are  probably  other  than  parasites. 

Gymnoasci. — Belonging  to  the  family  Gymnoasci  we  have  the 
genera  Trichophyton,  Microsporum  and  Achorion. 

The  trichophytons  are  generally  known  as  the  large-spored  ring- 
worms. The  spores  are  in  chains  and  may  be  inside  the  hair  or  both 


RINGWORM. 


'03 


outside  and  inside.     Many  of  them  are  of  animal  origin,  especially 

from  the  horse  and  the  cat. 

T.  Tonsurans. — Gives  a  crater-like  culture  with  fine  marginal 
rays.  Fungus  wholly  inside  the  hair.  Causes  most 
of  the  large-spored  scalp  ringworms  and  many 
body  cases. 


FIG.  41. — More  common  fungi,  i,  culture  of  Achorion  schoenleini  (favus); 
2  culture  of  Trichophyton  tonsurans;  3,  culture  of  Trichophyton  sabouraudi; 
4,  sporangium  of  Aspergillus;  5,  culture  of  Trichophyton  mentagrophytes; 
6,  culture  of  Microsporum  audouini;  7,  mycelium  and  spores  of  Malassezia  fur- 
fur; 8,  Cryptococcus  gilchristi;  9,  A  and  B,  sporangium  and  mycelium  of  Mucor 
corymbifer;  10,  Penicilium;  n,  Saccharomycestumefaciens;  12,  Discomyces  bovis. 


T.  Sabouraudi. — Has  a  heaped-up  festooned  sort  of  culture. 
There  is  a  similar  fungus  with  a  violet  culture. 
These  cause  some  of  the  scalp  and  beard  ring- 
worms. 

T.  Mentagrophytes. — This     is     the     megalosporon     endo- 


104  STUDY    AND    IDENTIFICATION    OF    MOULDS 

ectothrix  of  Sabouraud.  The  external  spores  are 
in  chains  or  in  short  mycelial  threads,  not  mosaics  of 
spores.  There  are  varieties  from  horse,  cat  and 
bird.  The  lesions  are  more  inflammatory  than 
those  of  the  endothrix  class.  Most  of  the  beard  and 
bcdy  ringworms  belong  to  this  group — very  few 
scalp  cases.  The  cultures  are  finely  rayed.  The 
so  called  small  spored  ringwrorm  is  the  Microsporum 
audouini.  The  fungus  is  packed  as  a  mcsaic  of 
spores,  chiefly  on  the  outside  of  the  hairs.  It  is 
the  chief  cause  of  the  ringworm  of  the  scalp  of 
children.  It  gives  a  downy-white  culture. 

The  Achorion  schoenleini  is  the  cause  of  favus.  The  cultures  are 
rather  wrinkled.  It  is  characterized  by  the  scutulum  or  favus  cup. 

In  the  suborder  Carpoascus  wre  have  to  consider  the  family  Peri- 
sporiaceae.  In  this  family  the  asci  are  completely  enclosed  by  the 
investing  membrane,  the  perithecium.  When  this  rots  the  spores  are 
set  free.  There  are  two  genera  of  interest.  Penicillium  and  Asper- 
gillus. 

Penicillium. — While  Penicillium  does  at  times  form  perithecia,  yet 
they  characteristically  show  chains  of  spores.  The  common  P.  glaucum 
resembles  a  hand  with  terminal  beads. 

P.  Crustaceum. — Is  the  common  blue-green  mould.     It  has 

been  deemed  pathogenic  in  cases  of  chronic  catarrh 

of  the  eustachian  tube  and  in  gastric  hyperacidity. 

Aspergillus. — These   have   sterigmata   carrying  chains  of  spores. 

Of  the  pathogenic  Aspergilli  we  have: 

1.  A.  fumigatus. — This  has  been  considered  as  the  cause  of 

pellagra. 

2.  A.  repens. — This  has  been  found  in  the  auditory  canal 

and  may  produce  a  false  membrane. 

3.  A.   flavus. — This    has    been  found  in  the  discharges  of 

chronic  ear  diseases. 

4.  A.   concentricus. — Tnis    is   the   cause   of    an   important 

tropical   ringworm,   Tinea   imbricata.     The   scales 


TROPICAL   SKIN   DISEASES.  105 

are  dry,  like  pieces  of  tissue-paper.  There  are 
generally  about  four  rings  which  do  not  heal  in 
the  center.  General  appearance  is  that  of  watered 
silk.  There  are  no  inflammatory  lesion^.  Com- 
mon in  Malay  peninsula.  Also  found  in  some  parts 
of  the  Philippines  and  in  China.  Some  authorities 
consider  the  fungus  to  be  a  Trichophyton. 


FIG.  42. — Tropical  fungi,  i,  concentric  rings  of  Aspergillus  concentricus; 
2,  sporangium  of  A.  concentricus;  3,  Aspergillus  pictor;  4,  Microsporoides  minu- 
tissimus;  5,  Trichosporum  giganteum;  6,  black  granules  of  Madurella  mycetomi; 
7,  yellow  grains  of  Discomyces  madurae. 


5.  A  pictor.— This  is  the  cause  of  a  skin  affection  of  Central 
America.  In  the  affection  colored  spots  appear 
on  the  skin,  chiefly  on  face,  forearms  and  chest. 
The  disease  is  attended  with  a  mangy  odor.  Spots 
are  of  various  colors;  if  the  superficial  epithelium 


106  STUDY    AND    IDENTIFICATION    OF    MOULDS. 

is  affected  we  have  a  dark  violet  color.     Deeper 
involvement  gives  red  spots. 

Hyphomycetes. — In  this  order  are  grouped  certain  genera  which 

cannot  properly  be  assigned  to  any  of  the  other  orders. 

Discomyces  bovis.  This  is  the  well-known  ray  fungus,  the  cause  of 
actinomycosis.  In  man  it  is  at  times  found  in  chronic  sup- 
purative  conditions  attended  with  much  granulation  tissue. 
Such  pus  may  show7  small  yellow-gray  granules  about  the 
size  of  a  pin's  head.  When  spread  out  between  two  slides 
the  central  portion  shows  a  net-work  of  mycelium  with  bulbous 
thread-like  rays  going  to  the  periphery.  The  " clubs"  at  the 
periphery  are  degenerate  structures  and  do  not  stain  by  Gram. 
The  central  mycelium  is  Gram  positive.  This  mould  is 
essentially  an  anaerobe  and  should  be  cultivated  in  a  deep 
glucose  agar  stab.  It  may  also  be  cultivated  in  bouillon. 
In  this  it  grows  at  bottom.  Growth  is  dry  and  chalky. 
In  diagnosis  look  for  the  little  granules.  Curetting  of  the 
sinuses  may  give  the  "ray  fungus"  when  they  are  not  found 
free  in  the  pus. 

Discomyces  madurae.  This  is  a  ray  fungus  found  in  the  yellow  "fish- 
roe"  granules  of  madura  foot.  It  is  strictly  aerobic  in 
cultures,  thus  differing  from  actinomycosis.  For  diagnosis 
proceed  as  for  D.  bovis. 

Madurella  mycetomi.  This  is  the  cause  of  the  black  "gunpowder" 
granules  of  madura  foot.  It  is  a  mycelial  mass  with  rather 
oval  shaped  swollen  segments.  It  is  at  times  cultivable  on 
potato  and  agar  as  felted  masses  of  gray  growth,  which  later 
becomes  almost  black. 

Malassezia  furfur.  This  is  the  fungus  of  Tinea  versicolor.  It  is 
common  both  in  temperate  and  in  tropical  climates.  It  is 
characterized  by  dirty  yellow  spots  about  covered  parts 
of  the  body.  Scrapings  show  a  profusion  of  mycelial 
threads  and  interspersed  spores.  It  is  very  difficult  to 
cultivate. 

Microsporoides  minutissimus.     This  is  generally  considered  as  the 


DIAGNOSIS  AND    CULTIVATION   OF   FUNGI.  IOf 

cause  of  Erythrasma  or  dhobies  itch,  a  very  common  inter- 
trigo  of  the  tropics.  It  is  characterized  by  its  narrow 
mycelium  and  small  spores.  Various  fungi  are  found  in  this 
affection.  Castellani  considers  the  cause  of  dhobies  itch 
to  be  a  trichophyton.  T.  Cruris. 

Trichosporum  giganteum.  This  is  the  cause  of  a  disease  of  the  hairs, 
known  in  Columbia  as  "Piedra,"  so-called  from  the  small 
gritty- like  masses  along  the  length  of  the  hair.  These  spores 
are  arranged  like  mosaics  about  the  hair. 

DIAGNOSIS  OF  FUNGI. 

The  most  expeditious  way  to  examine  fungi  is  to  treat  the  scales 
or  hairs  with  a  10%  solution  of  caustic  potash  or  soda.  Then  crush 
between  two  slides;  heat  moderately  over  the  flame  and  examine. 

Tribondeau's  method  is  to  treat  the  scales  with  ether,  then  with 
alcohol  and  finally  with  water.  Next  put  the  sediment  (it  is  convenient 
to  use  a  centrifuge)  in  a  drop  of  caustic  soda  solution.  Cover  with  a 
cover-glass,  and  after  the  preparation  has  stood  about  an  hour  run 
glycerin  under  the  cover-glass. 

A  very  satisfactory  method  is  to  scrape  the  scales  with  a  small 
scalpel,  and  smear  out  the  material  so  obtained  in  a  loopful  of  white 
of  egg  or  blood-serum  on  a  glass  slide.  By  scraping  vigorously  the 
serum  may  be  obtained  from  the  patient.  After  the  smear  has  dried, 
treat  it  with  alcohol  and  ether  to  get  rid  of  the  fat.  It  may  then  be 
stained  with  Wright's  stain  or  by  Gram's  method.  The  ordinary 
Gram  method  may  be  used  or  the  decolorizing  may  be  done  with 
aniline  oil,  observing  the  decolorization  under  the  low  power  of  the 
microscope. 

Yeasts  are  best  examined  in  hanging  drop  on  the  plain  slide  with 
vaselin  cell,  as  given  under  Blood. 

CULTIVATION  OF  FUNGI. 

Moulds  grow  well  on  media  with  an  acid  reaction,  so  that  by 
adjusting  the  reaction  to  +  2  percent  or  even  higher,  we  permit  of  the 
growth  of  the  fungi,  but  inhibit  bacterial  development. 


IO8  STUDY    AND    IDENTIFICATION    OF    MOULDS. 

Glycerin  agar,  bread  paste  or  potato  media  are  all  suitable,  but  the 
best  medium  is  that  of  Sabouraud: 

Maltose,  4     grams. 

Peptone,  i     grams. 

Agar,  1.5  grams. 

Water,  100      c.c. 

Make  the  reaction  about  +  2. 

Before  inoculating  media  with  moulds,  some  recommend  placing 
the  material  in  60%  alcohol  for  one  or  two  hours  to  kill  the  bacteria. 
The  moulds  withstand  such  treatment. 

In  cultivating  moulds  it  is  best  to  use  small  Erlenmeyer  flasks, 
containing  about  one-fourth  of  an  inch  of  media  on  the  bottom,  for 
the  development  of  the  colonies.  In  order  to  separate  the  mould  we 
may  take  the  hair  or  scales  on  a  sterile  slide  and  cut  them  into  small 
fragments  with  a  sterile  knife.  Then  moisten  a  platinum  loop  from 
the  surface  of  an  agar  slant,  touch  a  fragment  with  the  loop,  and  when 
it  adheres  transfer  it  to  the  agar  slant.  Make  four  or  five  inoculations 
on  the  surface  and  from  suitable  growth,  after  four  to  seven  days, 
inoculate  the  medium  in  the  Erlenmeyer  flask. 

Plauth  recommends  receiving  the  mould  material  between  two 
sterile  glass  slides.  Seal  the  edges  of  the  slides  w;Lh  wax  and  place 
the  preparation  in  a  moist  chamber  for  four  to  seven  days.  From 
developing  fungus  growth  inoculate  the  medium  in  the  Erlenmeyer 
flask.  A  Petri  dish  containing  several  layers  of  thoroughly  moistened 
filter-paper  in  top  and  bottom,  makes  a  satisfactory  moist  chamber. 


CHAPTER  XL 
BACTERIOLOGY  OF  WATER,  AIR,  MILK,  ETC. 

BACTERIOLOGICAL  EXAMINATION  OF  WATER. 

WHILE  in  a  chemical  examination  as  to  the  character  of  a  water 
there  are  certain  relations  between  the  ammonias,  nitrates,  chlorides,  etc., 
which  indicate  the  probable  animal  as  against  vegetable  nature  of  the 
organic  matter  present,  yet  it  is  a  more  or  less  presumptive  evidence. 
In  a  bacteriological  examination  of  water  the  finding  of  the  colon 
bacillus  may  from  a  practical  stand  point  be  considered  as  positive 
evidence  of  human  fecal  contamination.  Theoretically,  the  possibility 
of  organisms  being  present  corresponding  culturally  to  B.  coli  and  de- 
rived from  cereals  is  to  be  considered.  Also  the  faeces  of  animals  con- 
tain an  organism  which  cannot  be  differentiated  from  the  colon  bacillus. 

In  detecting  sewage  contamination  in  water  to  which  varying 
amounts  of  sewage  had  been  added,  it  was  found  that  the  bacterial 
tests  were  from  10  to  100  times  more  delicate  than  the  chemical  ones. 

As  showing  sewage  contamination  of  water,  the  presence  of  the 
B.  coli  has  been  generally  accepted  as  the  most  satisfactory  indication. 
The  English  authorities  consider  sewage  streptococci  and  the  spore- 
bearing  B.  enteritidis  sporogenes  as  of  value  as  indicators  as  well 
as  the  B.  coli — the  presence  of  sewage  streptococci  indicating  very 
recent  sewage  contamination  and  that  of  the  B.  enteritidis  sporogenes, 
in  the  absence  of  streptococci  and  colon  bacilli,  as  evidence  of  sewrage 
contamination  at  some  period  more  or  less  remote. 

In  the  United  States  the  colon  bacillus  alone  is  considered  the  indi- 
cator of  sewage  contamination,  and  all  tests,  presumptive  or  positive, 
are  based  on  the  presence  of  this  organism. 

In  collecting  samples  of  water  for  bacteriological  examination,  the 
following  points  should  be  considered: 

i.  The  bottles,  which  should  have  a  capacity  of  from  25  to  100  c.c,, 

109 


110  BACTERIOLOGY    OF    WATER,    AIR,    MILK,    ETC. 

should  be  sterile.  Sterilization  may  be  effected  by  heat  or  by  rinsing 
with  a  little  sulphuric  acid  and  subsequently  washing  out  thoroughly 
with  the  suspected  water  before  collection.  The  utmost  care  must 
be  exercised  that  the  ringers  do  not  come  in  contact  with  the  glass 
stopper  or  the  neck  of  the  bottle  while  filling  it.  If  the  specimen  is  to  be 
sent  some  distance,  it  should  be  packed  in  ice  to  prevent  bacterial 
development.  Frankland  states  that  a  count  of  1000  became  6000  in 
6  hours  and  48,000  in  48  hours.  In  water  packed  in  ice  for  a  consider- 
able time,  however,  the  bacterial  count  may  diminish. 

2.  If  collecting  from  city  water  supplies,  secure  the  sample  direct 
from  the  mains  and  let  the  water  run  from  the  tap  a  few  minutes  be- 
fore collection.  If  the  water  be  taken  from  a  pond,  stream  or  cistern, 
be  sure  that  the  specimen  comes  from  at  least  10  inches  below  the  sur- 
face. As  sedimentation  is  the  most  important  method  in  self-purifica- 
tion of  rivers  and  ponds,  it  will  be  understood  that  any  stirring  up  of 
the  mud  on  the  bottom  will  enormously  increase  a  bacterial  count. 

Quantitative  Bacteriological  Examination. 

i.  Deliver  definite  quantities  of  the  water  to  be  examined  into 
tubes  of  liquefied  gelatin  or  agar  and  plate  out  the  same  or  into  a 
series  of  Petri  dishes.  The  water  should  be  deposited  in  the  center 
of  the  plate  and  the  melted  gelatin  or  agar  poured  directly  on  the 
water  and  then,  carefully  tilting  to  and  fro,  mix  the  water  and  the 
media.  One  set  of  plates  should  be  of  gelatin  and  incubated  at 
room  temperature;  a  similar  set  should  be  of  lactose  litmus  agar  and 
incubated  at  38°  C.  If  the  water  is  highly  contaminated,  it  is  neces- 
sary to  dilute  it;  thus,  with  river  water,  which  may  contain  from  2000 
to  10,000  bacteria  per  c.c.,  a  dilution  of  i  to  100  would  be  desirable. 

Ordinarily  it  wrill  be  sufficient  to  deliver  from  a  sterile  graduated 
pipette  .2,  .3,  and  .5  c.c.  of  the  water  in  each  of  2  sets  of  plates:  one  set 
for  gelatin,  the  other  for  agar. 

When  gelatin  is  not  at  hand  or  convenient  to  work  with,  the  gelatin 
plates  may  be  replaced  by  others  of  lactose  litmus  agar  for  incubation 
at  room  temperature.  After  24  hours  at  38°  C.  or  48  hours  at  20°  C., 
the  count  should  be  made. 

Example:     Forty  colonies  were  counted  on  the  gelatin  plate  con- 


BACTERIOLOGY    OF    WATER.  Ill 

tainkig  .2  c.c.  (1/5)  of  the  water.  The  number  of  organisms  would  be 
200  per  c.c.  Ten  colonies  were  counted  on  the  agar  plate  containing 
.2  c.c.  and  incubated  at  38°  C.  Number  of  bacteria  developing  at  body 
temperature  equals  50  per  c.c. 

There  is  no  strict  standard  as  to  the  number  of  bacteria  a  water 
should  contain  per  c.c.  Koch's  standard  of  100  colonies  per  c.c.  is 
generally  given.  It  is  by  the  qualitative  rather  than  the  quantitative 
analysis  that  one  should  judge  a  water. 

If  there  should  be  very  many  colonies  on  a  plate,  the  surface  can  be 
marked  off  into  segments  with  a  blue  pencil.  If  very  numerous,  cut 
out  of  a  piece  of  paper  a  space  equal  to  i  square  centimeter.  By 
counting  the  number  of  colonies  inclosed  in  this  space  at  different 
parts  of  the  plate,  we  can  strike  an  average  for  each  space  of  i  square 
centimeter.  To  find  the  number  of  such  spaces  contained  in  the  plate, 
multiply  the  square  of  the  radius  of  the  plate  by  3.1416.  Then  multi- 
ply this  number  by  the  average  per  square  centimeter,  and  we  have  the 
total  number  of  colonies  on  the  plate.  This  is  the  principle  of  the 
Jeffers  disk. 

The  relative  proportion  between  the  bacterial  count  at  20°  C.  and 
that  at  38°  C.  is  of  great  importance  from  a  qualitative  stand-point,  as 
will  be  seen  later. 

2.  Deliver  into  a. series  of  Durham  fermentation  tubes  containing 
glucose  bouillon  and  into  another  series  containing  lactose  bouillon 
varying  definite  amounts  of  the  water  to  be  examined.  In  tubes  show- 
ing the  presence  of  gas  in  both  glucose  and  lactose  bouillon  the  evidence 
is  presumptive  that  the  colon  bacillus  is  present.  For  the  positive 
demonstration  plates  must  be  made  from  such  tubes  as  show  gas. 

It  is  sufficient  to  deliver  from  graduated  pipettes  in  each  series 
quantities  of  water  varying  in  amount  from  .1  c.c.  to  10  c.c.  In  our 
laboratory  we  inoculate  with  .1  c.c.,  .2  c.c.,  .5  c.c.,  i  c.c.  and  10  c.c. 
of  the  suspected  water.  If  the  .1  c.c.  tubes  show  gas,  we  have  reason 
to  assume  that  the  water  contained  at  least  10  colon  bacilli  per  c.c.  If 
only  the  10  c.c.  tubes  showed  gas — those  with  less  amounts  not  having 
gas — we  would  be  in  a  position  to  state  that  the  water  contained  the 
colon  bacillus  in  quantities  of  10  c.c.,  but  not  in  quantities  of  i  c.c.  or 
less  Many  authorities  regard  water  as  suspicious  only  when  the  colon 


112  BACTERIOLOGY    OF    WATER.    AIR,    MILK.    ETC. 

bacillus  is  present  in  quantities  of  10  c.c.  or  less;  waters  of  good  quali- 
ties frequently  showing  the  presence  of  the  colon  bacillus  in  quantities 
of  100  to  500  c.c. 

It  is  generally  accepted  that  if  a  water  shows  the  presence  of  the 
colon  bacillus  in  quantities  of  i  c.c.  or  less,  it  should  be  regarded  as 
suspicious. 

At  the  present  time  the  medium  that  gives  the  least  source  of  error 
in  carrying  out  the  quantitative  presumptive  tests  is  the  bile  lactose. 
It  is  made  by  adding  i%,  of  lactose  to  ox  bile,  and  fermentation  tubes 
of  the  media  showing  gas  may  be  considered  as  very  probably  con- 
taining the  colon  bacillus.  The  percentage  of  error  with  this  method 
is  reported  to  be  only  11%,  while  with  glucose  fermentation  tubes  the 
error  is  more  than  50%.  Gas  formation  is  usually  shown  in  48  hours, 
but  it  is  advisable  to  continue  the  incubation  for  72  hours.  Even  with 
this  method  plates  should  be  made. 

3 .  As  the  colon  and  sewage  streptococci  ferment  lactose  with  the  pro- 
duction of  acid  and  hence  produce  pink  colonies  on  lactose  litmus  agar, 
much  information  can  be  obtained  from  the  proportion  existing 
between  the  number  of  pink  colonies  and  those  not  having  such  a  color. 
Waters  of  fair  degree  of  purity  rarely  give  any  pink  colonies. 

Qualitative  Bacteriological  Examination. 

General  Considerations. — In  some  countries  the  proportion  of 
liquefying  to  nonliquefying  colonies  on  gelatin  plates  is  considered  of 
importance.  Certain  sewage  organisms  belonging  to  the  proteus  and 
cloaca  group  liquefy  gelatin;  consequently,  if  the  proportion  of  liquefy- 
ing to  nonliquefying  be  greater  than  as  i  to  10,  the  water  is  considered 
suspicious.  The  test  is  not  considered  by  American  authorities  as  of 
any  particular  value. 

The  American  Public  Health  Association  recognizes  the  importance 
of  the  information  obtained  from  a  comparison  of  the  number  of 
organisms  developing  at  38°  C.  and  those  developing  at  20°  C.  Bacteria 
whose  normal  habitat  is  the  intestinal  canal  naturally  develop  well  at 
body  temperature,  while  normal  water  bacteria  prefer  the  average 
temperature  of  the  water  in  rivers  and  lakes.  Consequently  when  the 


COLON    BACILLUS   IN   WATER  ANALYSIS.  113 

number  of  organisms  developing  at  38°  C.  at  all  approximates  the  num- 
ber developing  at  20°  C.,  there  is  a  strong  suspicion  that  sewage  or- 
ganisms may  be  present.  Normal  waters  give  proportions  of  i  to  25 
or  i  to  50,  while  in  sewage  contaminated  waters  the  proportion  may  be 
as  i  to  4  or  less. 

In  addition,  the  appearance  of  pink  colonies  on  the  lactose  litmus 
agar  is  of  great  assistance  in  judging  of  a  water.  Both  sewage  strepto- 
cocci and  the  colon  bacillus  give  pink  colonies — those  of  the  streptococci 
are  smaller  and  more  vermilion  in  color.  Microscopic  examination 
will  differentiate  the  cocci  from  the  bacilli.  It  is  well  to  bear  in  mind 
that  the  pink  colonies  after  24  hours  may  turn  blue  in  48  hours  from 
the  development  of  ammonia  and  amines.  Consequently  the  lactose 
litmus  agar  plates  should  be  studied  after  24  hours. 

A  good  water  supply  will  rarely  show  a  pink  colony,  while  in  a 
sewage  contaminated  one  the  pink  colonies  wrill  probably  predominate. 

The  diagnostic  characteristics  considered  important  by  the  Ameri- 
can authorities  in  reporting  the  colon  bacillus  are: 

1 .  Typical  morphology,  nonsporing  bacillus,  relatively  small  and 
often  quite  thick. 

2.  Motility  in  young  broth  cultures.  (This  is  at  times  unsatisfac- 
tory, as  some  strains  of  the  colon  bacillus  do  not  show  it  even  in  young 
bouillon  cultures.) 

3.  Gas  formula  in  dextrose  broth.     Of  about  50%  of  gas  produced, 
1/3  should  be  absorbed  by  a  2%  solution  of  sodium  hydrate  (CO2).   The 
remaining  gas  is  hydrogen.     (Later  views  indicate  that  the  gas  formula 
is  exceedingly  variable  and  should  not  be  depended  upon.     To  carry 
out  this  test  one  fills  the  bulb  of  a  fermentation  tube  with  the  caustic 
soda  solution  then,  holding  the  thumb  over  the  opening  or  with  a 
rubber  stopper,  the  bouillon  culture  and  the  soda  solution  are  mixed  by 
tilting  the  fermentation  tube  to  and  fro.     The  total  amount  of  gas  is 
first  recorded  and  then  that  remaining  after  the  CO2  has  been  absorbed 
is  reported  as  hydrogen.) 

4.  Nonliquefaction  of  gelatin. 

5.  Fermentation  of  lactose  with  gas  production. 

6.  Indol  production. 

7.  Reduction  of  nitrates  to  nitrites. 
8 


114  BACTERIOLOGY    OF    WATER,    AIR,    MILK,    ETC 

NOTE. — The  reduction  of  neutral  red  with  a  greenish-yellow 
fluorescence  is  very  striking  and  has  been  suggested  as  a  test  for  the 
colon  bacillus.  Many  other  organisms,  especially  those  of  the  hog 
cholera  group,  have  this  power.  It  is  convenient,  however,  to  color 
glucose  bouillon  with  about  i%  of  a  1/2%  solution  of  neutral  red. 

Isolation  of  the  Typhoid  Bacillus  from  Water. 

This  is  probably  the  most  discouraging  procedure  which  can  be 
taken  up  in  a  laboratory.  Only  the  most  recent  reports  of  such  isola- 
tion from  water  supplies,  which  have  been  verified  by  immunity  reac- 
tions, can  be  accepted  and  of  these  the  number  of  instances  is  exceed- 
ingly small.  Owing  to  the  long  period  of  incubation,  the  typhoid 
organisms  may  have  died  out  before  the  outbreak  of  an  epidemic 
suggests  the  examination  of  the  water  supply. 

There  have  been  various  methods  proposed  for  the  detection  of 
the  B.  typhosus  in  water.  A  method  which  wrould  offer  about  as 
reasonable  a  chance  of  success  as  any  other  would  be  to  pass  2  or  3 
liters  of  the  water  through  a  Berkefeld  filter;  then  to  take  up  in  a 
small  quantity  of  water  all  the  bacteria  held  back  by  the  filter.  Then 
plate  oat  on  lactose  litmus  agar  and  examine  colonies  which  do  not 
show  any  pink  coloration.  The  dysentery  bacillus  has  about  the  same 
cultural  characteristics  as  the  typhoid  one,  so  that  it  is  important  to 
note  motility.  If  from  such  a  colony  you  obtain  an  organism  giving 
the  cultural  characteristics  of  B.  typhosus,  carry  out  agglutination  and 
preferably  bacteriolytic  tests  as  well.  Some  strains  of  typhoid,  espe- 
cially when  recently  isolated  from  the  body,  do  not  show  agglutination. 

The  Conradi  Drigalski,  the  malachite-green  and  various  caffeine 
containing  plating  media  have  been  highly  recommended. 

Isolation  of  the  Cholera  Spirillum  from  Water. 

The  method  proposed  by  Koch  in  1893  does  not  seem  to  have  been 
improved  upon  by  later  investigators.  To  100  c.c.  of  the  suspected 
water  add  i%  of  peptone  and  i%  of  salt.  Incubate  at  38°  C.,  and  at 
intervals  of  8,  12  and  18  hours  examine  microscopically  loopfuls  taken 
from  the  surface  of  the  liquid  in  the  flask.  So  soon  as  comma-shape 


BACTERIOLOGICAL   EXAMINATION   OF   MILK.  1 15 

organisms  are  observed,  plate  out  on  agar.  The  colonies  showing 
morphologically  characteristic  organisms  should  be  tested  as  to  ag- 
glutination and  bacteriolysis.  Inasmuch  as  the  true  cholera  spirillum 
shows  a  marked  cholera-red  reaction  it  is  well  to  inoculate  a  tube  of 
peptone  solution  from  such  a  colony  and  add  a  drop  of  concentrated 
sulphuric  acid  after  incubating  for  18  hours.  The  rose-pink  colora- 
tion is  given  by  the  cholera  spirillum  with  the  acid  alone — the  nitroso 
factor  in  the  reaction  being  produced  by  the  organism. 

BACTERIOLOGICAL  EXAMINATION  OF  MILK. 

A  bacterial  milk  count  is  of  comparatively  little  value  as  showing 
whether  a  milk  is  dangerous  or  not.  As  a  matter  of  fact,  a  milk  which 
contains  several  million  of  bacteria  per  c.c.  might  be  less  dangerous 
than  one  containing  only  a  few  thousand,  especially  if  in  the  latter 
there  were  numerous  liquefiers  and  gas  producers  present.  There  is, 
however,  one  point  of  importance  in  connection  with  the  quantitative 
estimation  of  bacteria  in  milk,  and  that  is  the  fact  that  in  order  to  keep 
the  development  of  the  bacteria  within  the  limits  of  10,000  to  50,000 
per  c.c.,  it  is  necessary  that  the  requirements  of  cleanliness  in  milking 
and  the  rapid  cooling  of  the  milk  after  obtaining  it  and  the  keeping 
of  the  temperature  below  50°  C.  be  rigidly  observed.  If  a  milk  has  a 
high  count  it  shows  some  error  in  the  handling  of  the  milk.  In  making 
a  quantitative  bacteriological  examination,  the  principle  is  the  same 
as  with  water. 

Make  a  known  dilution  of  the  milk  with  sterile  water;  add  definite 
quantities  of  this  diluted  milk  to  tubes  of  melted  agar  or  gelatin  and 
pour  into  plates.  The  diluted  milk  may  also  be  delivered  in  the  center 
of  the  plate  and  the  melted  agar  or  gelatin  poured  directly  on  it, 
mixing  thoroughly.  .  Always  shake  the  bottle  well  before  taking 
sample. 

Example:  Added  i  c.c.  of  milk  to  199  c.c.  of  sterile  water  in  a 
large  flask  (500  to  1000  c.c.).  After  shaking  thoroughly,  take  i  c.c. 
of  this  i :  200  dilution  and  add  it  to  99  c.c.  of  sterile  water.  Shak- 
ing thoroughly,  wre  have  a  dilution  of  i :  20,000.  Of  this  we  added 
.5  c.c.  to  a  tube  of  gelatin  or  agar.  After  incubation  the  plate  showed 


Il6  BACTERIOLOGY    OF    WATER,    AIR,    MILK;    ETC. 

75  colonies.  Therefore  the  milk  contained  in  each  c.c.  75  x  2  x  20,000 
(dilution)  =  3,000,000 — the  number  of  bacteria  in  each  c.c.  of  milk. 

Lactose  litmus  gelatin  or  agar  is  to  be  preferred  in  milk-work,  as 
the  normal  lactic  acid  bacteria  produce  reddish  colonies  which  are  very 
striking.  A  standard  easily  attained  for  high-grade,  certified  milk 
would  be  5,000  to  10,000  per  c.c. 

In  the  qualitative  examination  of  milk,  many  dairies  employ  the 
fermentation  tube,  any  organism  producing  gas  being  considered 
undesirable.  Again  liquefying  organisms,  as  shown  by  the  presence 
of  such  bacteria  in  the  gelatin  plates,  is  evidence  of  probable  contami- 
nation by  fecal  bacteria.  A  question  which  seems  difficult  to  decide 
is  as  to  the  general  nature  of  the  so-called  normal  lactic  acid  bacteria 
of  milk.  Some  describe  them  as  very  short,  broad  bacilli  with  very 
small  colonies,  fermenting  lactose  with  the  formation  of  lactic  acid. 
Others  consider  that  the  streptococci  are  the  organisms  which  are 
concerned  with  the  normal  fermentative  changes.  In  examining 
specimens  of  milk  considered  the  best  on  the  market,  I  have  repeatedly 
found  the  small  red  colonies  on  lactose  litmus  agar  to  be  streptococci. 
In  connection  with  the  organisms  present  in  the  tablets  used  for  treat- 
ing milk  to  produce  lactic  acid  for  the  treatment  of  intestinal  disorders, 
and  considered  to  be  normal  lactic  acid  bacteria,  I  have  found  both 
streptococci  and  bacilli.  These  have  all  agreed,  however,  in  not  pro- 
ducing gas  in  either  lactose  or  glucose  fermentation  tubes. 

Another  source  of  information  as  to  the  quality  of  a  milk  may  be 
derived  from  a  study  of  the  number  of  leukocytes  or  pus  cells  con- 
tained in  i  c.c.  of  the  milk. 

The  Doane-Buckley  method  is  probably  the  most  accurate.  In 
this  you  throw  down  the  cellular  contents  of  10  c.c.  of  milk  in  a  cen- 
trifuge revolving  about  1000  times  a  minute  for  ten  to  twenty  minutes. 
Then  remove  supernatant  milk  and  add  0.5  c.c.  of  Toisson's  solution 
to  the  sediment.  You  thus  have  the  leukocytes  of  10  c.c.  contained  in 
0.5  c.c.  (Concentrated  twenty  times.)  Make  a  haemacytometer  pre- 
paration as  for  blood  and  find  the  average  number  of  cells  for  each 
square  millimeter.  Then  multiply  this  by  10  to  get  the  number  of 
cells  in  a  cubic  millimeter.  As  a  cubic  millimeter  is  one  thousand 
times  smaller  than  a  cubic  centimeter,  you  multiply  the  number  per 


BACTERIOLOGICAL   EXAMINATION   OF  AIR.  Ilj 

cubic  millimeter  by  one  thousand.  Then,  as  the  milk  was  concentrated 
twenty  times,  you  divide  by  20.  (If  it  were  diluted  twenty  times, 
you  would  multiply  by  20.) 

Example:  Found  an  average  of  50  cells  per  square  millimeter. 
This  would  make  500  per  cubic  millimeter,  and  500,000  per  c.c.; 
then  500,000  divided  by  20  would  give  25,000. 

There  is  no  agreement  as  to  a  standard  for  allowable  leukocytes. 
Even  in  apparently  healthy  animals  they  may  exceed  100,000  per  c.c. 
Doane  has  suggested  500,000  per  c.c.  as  a  preferable  limit. 

The  smear  methods  for  determining  the  number  of  leukocytes 
present  do  not  compare  in  accuracy  with  the  volumetric  ones. 

BACTERIOLOGICAL  EXAMINATION  OF  AIR. 

In  Paris  a  cubic  meter  of  air  was  found  to  contain  the  following 
number  of  organisms: 

Suburbs.— Winter,       145  moulds,  170  bacteria. 

Summer,      245  moulds,      345  bacteria. 

City  Hall. — Winter,     1345  moulds,  4305  bacteria. 

Summer,  2500  moulds,  9845  bacteria. 

Air  of  hospitals,  especially  after  sweeping,  may  contain  50,000 
bacteria  per  cubic  meter.  There  does  not  seem  to  be  any  particular 
relation  between  the  amount  of  carbon  dioxide  in  air  and  the  bacterial 
content. 

Petri's  Rough  Method. — Exposure  of  a  lactose  litmus agar  plate 
(capacity  100  sq.  cm.)  for  five  minutes  will  give  the  number  of  organ- 
isms present  in  ten  liters  of  air.  Multiply  by  100  for  one  cubic  meter. 

The  two  groups  of  organisms  usually  found  in  air  are  (i)  bacteria 
and  (2)  moulds.  Moulds  (spores)  may  be  carried  by  currents  of  air; 
bacteria,  however,  are  generally  carried  about  by  particles  of  dust  or 
finely  divided  liquids  (spray).  On  the  lactose  litmus  agar  plate 
staphylococci  and  streptococci  show  as  bright  red  colonies. 

Sedgwick  Tucker  Sterile  Granulated  Sugar  Method. — Sterilize 
aerobioscope  and  introduce  granulated  sugar  on  support.  Again 
sterilize  (not  over  120°  C.  in  dry-air  sterilizer).  Allow  a  given  quan- 
tity of  air  to  pass  through;  then  shake  the  sugar  into  wide  part  of 


Il8  BACTERIOLOGY    OF    WATER,    AIR,    MILK,    ETC. 

aerobioscope.  Now  pour  in  10  or  15  c.c.  of  melted  gelatin  (40°  C.)  to 
dissolve  sugar.  Roll  tubes  as  for  Esmarch  roll  cultures,  and  incubate 
at  room  temperature.  To  draw  air  through  the  aerobioscope,  connect 
the  small  end  with  a  piece  of  rubber  tubing  which  is  attached  to  a  tube 
in  the  stopper  of  an  aspirating  bottle.  Having  poured  a  definite 
quantity  of  wrater  into  the  aspirating  bottle,  allow  the  water  to  run  out. 
The  same  quantity  of  air  will  be  drawn  through  the  sugar  of  the 
aerobioscope  as  the  amount  of  water  passing  out  of  the  aspirating 
bottle.  The  bacteria  and  moulds  are  caught  by  the  sugar. 

Example. — Passed  10  liters  of  air  through  the  aerobioscope.     The 
bacteria  in  this  quantity  of  air  showed  75  colonies  when  incubated 


FIG.  43. — Sedgwick-Tucker  aerobioscope.     (Williams.} 

at  20°  C.  The  unit  being  one  cubic  meter  or  one  thousand  liters, 
we  have  only  obtained  the  bacteria  of  one  hundredth  of  the  unit. 
Hence  multiplying  75  by  100  gives  7,500  bacteria  as  present  in  one 
cubic  meter  of  the  air  examined. 

In  comparing  the  results  with  the  aerobioscope  with  those  obtained 
by  exposing  a  plate  as  in  Petri's  method  for  ten  instead  of  five  minutes, 
it  was  found  that  the  latter  was  sufficiently  in  accord  to  make  it  a 
satisfactory  approximate  quantitative  method.  The  simplicity  and 
ease  of  access  of  the  colonies  developing  in  it  make  it  preferable  when 
the  air  of  operating-rooms  or  hospital  wards  is  to  be  examined. 


CHAPTER  XII. 
PRACTICAL  METHODS  IN  IMMUNITY. 

THAT  which  prevents  the  gaining  of  a  foothold  by  disease  organisms 
in  the  animal  body  or  which  neutralizes  their  harmful  products  or 
destroys  the  parasites  is  termed  immunity.  In  the  main,  the  question 
of  immunity  hinges  on  the  powers  of  resistance  of  the  human  body 
and  the  aggressiveness  or  virulence  of  the  invading  organism.  It 
must  always  be  kept  in  mind  that  immunity  is  only  relative;  thus  the 
fowl,  which  is  practically  immune  to  tetanus,  may  be  made  to  suc- 
cumb by  reducing  its  resistance  by  refrigeration  or  by  increasing  the 
amount  of  poison  introduced.  The  insusceptibility  which  the  fowl 
has  to  tetanus  or  which  man  has  to  many  diseases  of  animals  is  best 
termed  inherent  immunity,  and  is  at  present  only  a  subject  of  theo- 
retical interest.  When  immunity  to  a  given  disease  is  obtained  as  a 
result  of  an  attack  of  the  disease  in  question  or  by  laboratory  methods 
of  inoculation,  this  is  termed  properly  an  acquired  immunity,  and  in 
the  former  case  is  a  naturally  acquired  immunity  or  "natural  im- 
munity" and  in  the  second  is  an  artificially  acquired  immunity  or 
" artificial  immunity." 

As  a  result  of  an  attack  of  a  disease  or  in  response  to  the  stimulus 
of  the  injection  of  the  organism  or  its  products,  we  have  developed  in 
the  man  so  injected  certain  specific  antagonistic  properties  to  that 
organism,  which  are  usually  demonstrable  in  the  blood  serum  or 
other  body  fluids,  and  to  which  we  apply  the  terms  agglutinating 
power,  opsonic  power  or  bacteriolytic  power.  The  term  antibody  is 
also  applied.  All  three  powers  may  be  present  together  in  equal  or  in 
varying  degree  or  one  or  more  may  be  absent.  By  agglutinating 
power  we  mean  that  which  causes  evenly  distributed  organisms  to 
come  together  and  form  clumps.  By  opsonic  power  we  mean  that 
which  so  alters  the  resistance  of  bacteria  that  the  phagocytes  ingest 
them.  By  bacteriolytic  power  we  mean  that  which  brings  about 

119 


120 


PRACTICAL    METHODS    IN    IMMUNITY. 


disintegration  or  lysis  of  the  specific  organism.  The  bacterium 
which  causes  the  disease  or  which  is  used  in  inoculation  for  the  pro- 
duction of  immunity  is  termed  the  specific  organism. 

Of  the  different  kinds  of  immunity  only  artificial  immunity  will 
be  considered.  This  may  be  obtained  in  two  ways :  i .  By  injecting  the 
bacteria  or  their  products  into  man  or  animals  and  as  the  result  of 
the  activity  of  the  cells  of  the  animal  invaded,  antibodies  are  formed 

which  neutralize  the  toxins  of 
or  destroy  the  specific  bac- 
teria. These  antibodies  which 
are  supposed  to  be  thrown  off 
(free  receptors)  or  which  may 
remain  attached  to  the  cell 
(sessile  receptors)  may  re- 
main potential  for  months  or 
years  and  so  confer  a  more 
or  less  enduring  immunity. 
This  is  termed  active  im- 
munity. 2.  When  we  take 
the  serum  of  a  man  or  animal 
immunized  actively  and  inject 
it  with  its  contained  anti- 
bodies into  a  second  animal 
or  man,  we  confer  an  im- 
munity on  the  second  animal; 
but  as  his  cells  take  no  active 
part  in  the  production  of  the 
immunity,  but  are  only  pas- 
sive, we  term  this  immunity  " passive  immunity."  If  this  serum  which 
is  introduced  in  passive  immunity  only  neutralizes  the  toxic  products 
of  the  infecting  bacteria,  we  term  it  antitoxic  passive  immunity  and 
designate  the  immune  serum  as  antitoxic  serum.  If  it  destroys  the 
organism,  we  call  it  antimicrobic  serum,  and  the  immunity,  antimi- 
crobic  passive  immunity.  Some  immune  sera  are  both  antitoxic  and 
antimicrobic. 

It  is  well  to  remember  that  some  organisms  produce  a  toxin  which 


FIG.  44. — Receptors  of  the  first  order  uniting 
with  toxin.  (Journal  oj  the  American  Medi- 
cal Association.  1905.  P.  955.) 

a,  Cell  receptor;  6,  toxin  molecule;  c,  hap- 
tophore  of  the  toxin  molecule;  d,  toxophore 
of  the  toxin  molecule;  e,  haptophore  of  the 
cell  receptor. 


ANTITOXIC  AND  ANTIMICROBIC   SERA. 


121 


is  given  off  while  the  bacterium  is  alive;  and  in  other  instances  the  toxin 
is  intracellular  and  is  o  ily  given  off  when  the  bacterium  disintegrates; 
consequently,  an  antimicrobic  serum  may  cause  the  liberation  of  toxin. 
Diphtheria,  tetanus  or  botulism  antisera  are  instances  of  antitoxic 
sera,  while  practically  all  others  are  antimicrobic.  There  is  but  one 
factor  to  consider  in  an  antitoxic  serum  and  that  is  the  protoplasmic 
particles  which  are  thrown 
off  from  the  cell  in  response 
to  the  injury  incident  to  the 
attack  upon  the  cell  by  the 
toxin  particles.  This  free 
particle  in  the  circulation 
represents  the  entire 
mechanism  of  antitoxic  im- 
munity. It  is  capable  of 
uniting  with  the  toxin  mole- 
cule and  neutralizing  its 
toxic  power,  or  rather  so 
binding  its  combining  end 
(haptophore  group)  that  it 
is  incapable  of  attaching 
itself  to  a  cell,  so  that  the 
poisonous  end  of  the  toxin 
(toxophore  group)  cannot 
have  access  to  the  cell.  In 
antimicrobic  sera  we  have 
two  factors  to  consider,  the 
first  is  a  protoplasmic  par- 
ticle quite  similar  to  the  anti- 
toxin molecule,  but  which  in  itself  has  no  power  of  injuring  its  specific 
bacterium.  This  particle  is  generally  referred  to  as  the  amboceptor 
or  immune  body.  It  is  the  specific  product  of  the  activity  of  a  specific 
bacterium  or  foreign  cell  against  the  body  cells  attacked.  It  with- 
stands a  temperature  above  56°  C.  and  of  itself  is  incapable  of  injuring 
the  bacterium  in  response  to  whose  attack  it  was  produced.  The 
second  factor  in  the  bacteriolysis  of  the  specific  bacterium,  or  the 


FIG.  45. — Receptors  of  the  second  order  and 
of  some  substance  uniting  with  one  of  them. 
(Journal  of  the  American  Medical  Association. 
1905.  P.  1113.) 

c,  Cell  recepto:  of  the  second  order;  d,  tox- 
ophore or  zymophore  group  of  the  receptor; 
e ,  haptophore  of  the  receptor;  /,  Food  substance 
or  product  of  bacterial  disintegration  uniting 
with  the  haptophore  of  the  cell  receptor. 


122 


PRACTICAL   METHODS    IN    IMMUNITY. 


haemolysis  of  the  specific  foreign  cell,  is  something  normally  present 
in  the  serum  of  every  animal,  and  which  is  capable  of  disintegrating  a 
foreign  cell  or  bacterium,  provided  it  can  have  access  to  the  cell  or 
bacterium  through  an  intermediary  amboceptor  (hence  the  ambo- 
ceptor  is  sometimes  called  an  intermediary  body).  This  something 
is  called  the  "complement."  It  is  by  some  called  "alexine,"  by 
others  cytase  (Metchnikoff).  The  complement  cannot  act  upon  and 
destroy  an  invading  bacterium  or  cell  unless  the  amboceptor  is 


FIG.  46. — Receptor  of  the  third  order,  and  of  some  substance  uniting  with  one 
of  them.  (Journal  0}  the  American  Medical  Association.  1905.  P  1369  ) 

c,  Cell  receptor  of  the  third  order — an  amboceptor;  e,  one  of  the  haptophores  of 
the  amboceptor,  with  which  some  food  substance  or  product  of  bacterial  disintegra- 
tion (/)  may  unite;  g,  the  other  haptophore  of  the  amboceptor  with  which  com- 
plement may  unite;  k,  complement ;  h,  the  haptophore;  z,  the  zymotoxic  group  of 
complements. 


present  to  make  the  necessary  connection.  The  complement  is 
destroyed  by  a  temperature  of  56°  C.,  so  that,  if  we  heat  the  serum 
from  an  immune  animal  to  56°  C.,  the  complement  it  naturally  con- 
tains is  destroyed,  and  the  amboceptor  it  contains,  which  is  not 
injured  by  such  a  temperature,  is  incapable  of  destroying  bacteria  or 
cells,  unless  we  replace  the  complement  which  has  been  destroyed  by 
fresh  complement.  This  is  done  experimentally  by  adding  the  serum 


ACTIVATION    OF   IMMUNE    SERA. 


I23 


of  a  nonimmunized  animal  which  contains  the  complement,  but  no 
specific  immune  body  (amboceptor)  to  the  heated  serum.  This  is 
termed  "activating,"  and  a  serum  so  treated  is  said  to  be  "activated." 
When  an  immune  serum  has  been  heated  to  56°  C.,  it  is  said  to  have 
been  "inactivated". 


FIG.  47. — i,  Red  cells  +  normal  serum.  No  amboceptor.  Nohemolysis.  A.  com- 
plement; B,  normal  red  cell.  2.  Red  cells  +  immune  serum.  Complement  and 
amboceptor.  Hemolysis.  C,  complement;  D,  amboceptor;  E,  hemolyzed  red  cell. 

3.  Red  cells  +  immune  serum  heated  to  56°  C.     Inactivated.     Complement  de- 
strojed.     No  hemolysis,     F,  destroyed  complement;  G,  amboceptor;  H,  red  cells. 

4.  Red    cells  +  heated  immune  serum  +  fresh  serum.     (Activated  by  contained 
complement).    Hemolysis      I,  destroyed  complement;  J,  fresh  complement ;  K,  am- 
boceptor;   L,  hemolyzed   red   cell.     5,    Diagram   showing  antitoxin   production. 
a,  toxin  molecule;  &,  antitoxin  molecule;  c,  neutralization  of  toxin  by  antitoxin. 
6.  Diagram  showing  bacteriolysin.     d,  complement;  e,  amboceptor;  /,  bacillus. 


When  we  allow  a  mixture  of  bacteria  or  cells  to  remain  in  contact 
with  their  specific  immune  serum  which  has  been  inactivated,  the 
amboceptors  attach  themselves  to  the  bacteria  or  cells,  so  that  now,  upon 
adding  normal  serum  (complement),  these  bacteria  or  cells  are  so  pre- 


124  PRACTICAL    METHODS    IN    IMMUNITY. 

pared  that  the  complement  can  disintegrate  them.     This  experiment 
is  termed  " sensitizing"  and  cells  so  treated  are  said  to  be  "sensitized." 

METHODS  FOR  OBTAINING  IMMUNE  SERA. 

While  a  convalescent  from  a  disease  may  be  utilized  to  obtain  an 
antitoxic,  agglutinating,  opsonic  or  bacteriolytic  serum  against  the 
specific  bacterium,  yet  this  is  more  conveniently  obtained  from  an 
animal  which  has  been  immunized  against  the  bacterium  or  cell  in 
question.  The  rabbit  is  the  most  convenient  animal  to  employ  for  the 
production  of  immune  sera  where  the  object  is  to  have  at  hand  a  serum 
for  use  in  diagnosis. 

Where  sera  are  used  on  an  extensive  scale,  as  in  the  production  of 
curative  sera,  larger  animals  are  employed.  There  are  two  application 
of  serum  diagnosis :  i .  Where  the  bacterium  is  known  and  the  serum 
is  to  be  diagnosed.  2.  Where  the  serum  is  known  and  the  bacterium 
is  to  be  diagnosed. 

The  first  is  employed  by  testing  the  agglutinating  or  bacteriolytic 
power  of  the  serum  taken  from  a  patient  upon  pure  cultures  of  the  organ- 
ism which  is  suspected  as  the  cause  of  the  disease.  The  Widal  test  (ag- 
glutination) is  the  best  instance  of  this  procedure.  This  method  is 
of  practical  value  in  the  diagnosis  only  of  typhoid,  Malta  fever  and 
paratyphoid.  In  diseases  like  cholera  and  bacillary  dysentery,  the 
disease  has  run  its  course  before  agglutinating  power  becomes  apparent 
in  the  serum.  This  method,  however,  may  be  used  to  prove  that 
a  convalescent  has  suffered  from  a  suspected  disease.  Thus,  by  test- 
ing the  agglutinating  power  of  a  serum,  one  or  two  weeks  after  re- 
covery from  a  suspicious  case  of  ptomaine  poisoning,  we  may  be  able 
to  demonstrate  that  the  case  in  question  was  cholera.  The  second 
method  has  wider  application,  and  is  the  one  in  which  we  use  the  sera 
of  animals  which  have  been  immunized  with  known  bacteria.  Or- 
ganisms isolated  from  urine,  faeces  or  blood  of  patients,  or  those  obtained 
from  water  or  food  supplies  may  be  identified  by  testing  the  agglu- 
tinating, opsonic  or  bacteriolytic  power  of  known  sera  against  them. 
This  has  a  wide  range  of  applicability.  The  testing  of  the  opsonic 
power  of  the  sera  in  man  or  animals  immunized  against  plague,  and 
possibly  cerebrospinal  meningitis,  seems  to  give  more  definite  informa- 


METHODS   FOR   OBTAINING   IMMUNE    SERA.  125 

tion  than  do  agglutination  or  bacteriolytic  tests.  With  the  majority 
of  other  organisms,  however,  the  agglutination  test  is  the  one  almost 
always  preferred. 

Even  in  a  small  laboratory  there  are  no  particular  difficulties  in  the 
way  of  having  on  hand  rabbits  immunized  against  typhoid,  paratyphoid 
Malta  fever,  acid  producing  and  nonacid  producing  strains  of  dysentery, 
cholera,  etc.  Just  as  we  inject  men  with  vaccines  prepared  from 
various  bacteria  in  opsonic  therapy,  so  we  inject  animals  to  produce  sera 
for  diagnosis.  We  may  use  either  a  bouillon  culture  or  the  growth  on 
agar  slants  taken  up  with  salt  solution  as  the  inoculating  material. 
This  is  heated  for  one  hour  at  60°  C.  to  kill  the  bacteria.  Where  we 
desire  to  produce  a  serum  which  will  disintegrate  red  blood  cells 
(haemolytic  serum),  we  inject  about  4  c.c.  of  the  defibrinated  blood  of 
the  animal  for  which  we  wish  to  produce  a  specific  serum.  Thus  for 
a  serum  for  use  in  a  medicolegal  case  we  would  inject  the  rabbit  with 
human  blood.  The  most  convenient  way  to  defibrinate  blood  is  to 
break  a  section  of  glass  tubing  into  fragments,  put  these  fragments 
into  a  glass  test-tube,  sterilize  tube  and  contents  in  a  flame  or  sterilizer 
and,  when  cool,  let  the  blood  drop  into  the  test-tube  (we  may  use  a 
Wright's  pipette  with  a  rubber  bulb  to  take  up  the  blood  from  a  punc- 
ture of  the  finger  and  eject  it  into  the  tube).  By  shaking,  the  fibrin 
collects  on  the  glass  fragments,  and  we  have  the  corpuscular  emulsion 
to  inject.  Inject  about  4  c.c.  of  the  defibrinated  blood  or  i  c.c.  of  the 
killed  bacterial  bouillon  culture  into  the  peritoneal  cavity  of  the  rabbit. 
The  easiest  way  to  inject  the  rabbit  is  to  hold  the  animal  head  down 
and  plunge  the  needle  in  the  median  line  into  the  abdominal  cavity, 
forcing  in  the  contents  of  the  syringe.  The  intestines  gravitate  down- 
ward and  by  entering  the  needle  below  the  limits  of  the  bladder 
we  avoid  injuring  any  vital  part.  It  may  be  more  satisfactory  to  at 
first  inject  only  about  1/2  c.c.,  and  then  if  there  is  very  little  reaction, 
as  shown  by  the  appetite  and  spirits  of  the  rabbit,  to  inject  about  4 
days  latter  i  c.c.  About  4  or  5  injections  at  intervals  of  3  to  5  days 
will  usually  produce  an  immune  serum.  Some  animals  do  not  seem 
to  be  capable  of  producing  antibodies,  so  that  it  may  be  necessary  to 
use  one  or  more  rabbits  before  a  satisfactory  serum  is  obtained.  The 
most  convenient  way  of  obtaining  serum  for  a  test  is  to  cut  across  one 


126  PRACTICAL    METHODS    IN    IMMUNITY. 

of  the  marginal  veins  of  the  rabbit's  ear,  and  collect  the  blood  in  a 
Wright's  U-tube.  Centrifugalizing,  we  have  the  serum  ready  for  use. 
The  immune  body  and  agglutinin  in  serum  remain  active  for  weeks 
when  kept  in  the  refrigerator.  The  complement  and  opsonin,  however, 
begin  to  deteriorate  at  once  and  have  disappeared  by  the  fifth  day. 
Consequently,  for  opsonic  and  bacteriolytic  and  haemolytic  experiments, 
fresh  serum — 12  to  24  hours — must  be  used,  or  it  may  be  activated. 

AGGLUTINATION  TESTS. 

There  are  two  methods  of  testing  the  agglutinating  powers  of  a 
serum — the  microscopical  and  the  macroscopical  or  sedimentation 
method. 

i.  For  the  microscopical  method  draw  up  serum  to  the  mark  .5 
of  the  white  pipette.  Then  draw  up  salt  solution  to  the  mark  1 1 . 
This  when  mixed  gives  a  dilution  of  i  to  20.  One  loopful  of  the  diluted 
serum  and  one  loopful  of  a  bouillon  culture  or  salt  solution  suspen- 
sion of  the  organism  to  be  tested  gives  a  dilution  of  i  to  40.  One  loop- 
ful of  the  1-20  diluted  serum  and  3  loopfuls  of  the  bacterial  suspension 
give  a  dilution  of  1-80.  These  two  dilutions  answer  in  ordinary  diag- 
nostic tests.  The  red  pipette  with  a  i-ioo  or  1-200  dilution  may  be 
used  where  dilutions  approaching  i-iooo  are  desired.  Having  mixed 
the  diluted  serum  and  the  bacterial  suspension  on  a  cover-glass,  we 
invert  it  over  a  vaselined  concave  slide  and  examine  with  a  high 
power,  a  dry  objective  (1/6  in.).  It  is  simpler  to  make  a  ring  of  vaselin 
to  fit  the  cover-glass  and  make  the  mixture  of  diluted  serum  and  culture 
in  the  center  of  this  ring  or  square.  Then  apply  the  cover-glass,  press 
it  down  on  the  vaselin  ring  and  examine  as  with  the  ordinary  hanging 
drop.  In  making  dilutions  it  is  preferable  to  use  salt  solution,  as  the 
phenomenon  of  agglutination  requires  the  presence  of  salts.  Ordi- 
narily, 30  minutes  is  a  sufficient  time  to  wait  before  reporting  the 
absence  of  agglutination.  Agglutination  is  more  rapid  at  body  tempera- 
ture than  at  room  temperature.  In  reporting  agglutination,  always 
give  time  and  dilution.  It  is  absolutely  necessary  that  a  control  prepa- 
ration be  prepared  in  every  instance;  that  is,  one  with  the  bacterial 
culture  alone  or  with  a  normal  serum  of  the  same  dilution  as  the  lowest 
used.  Some  normal  sera  will  agglutinate  in  i  to  10  dilution,  and  group 


MACROSCOPICAL  AGGLUTINATION.  127 

agglutinations  (as  paratyphoid  with  typhoid  serum)  may  occur  in  i 
to  40  or  possibly  higher.  It  is  very  unusual  for  sera  to  agglutinate 
any  other  bacteria  than  its  specific  one  in  dilutions  as  high  as  1-80. 
2.  For  the  macroscopical  or  sedimentation  test,  take  a  series  of 
small  test-tubes  (3/8  x  3  in.)  and  deposit  i  c.c.  of  salt  solution  in  each 
of  the  series.  Now,  having  taken  an  empty  test-tube,  drop  4  drops  of 
serum  in  it  and  then  add  1 2  drops  of  salt  solution.  This  approximately 
gives  i  c.c.  of  a  1-4  dilution  of  the  serum.  With  a  rubber-bulb  capillary 
pipette,  which  has  been  graduated  to  hold  16  drops  or  i  c.c.  draw  up  the 
contents  of  the  tube  containing  the  i  to  4  serum  and  add  it  to  the  next 
tube  containing  i  c.c.  of  salt  solution.  This  gives  a  dilution  of  i  to  8. 
Now  mix  thoroughly  by  drawing  up  and  forcing  out  with  the  bulb 
pipette,  and  then  withdraw  i  c.c  and  add  to  the  next  tube  containing 
i  c.c.  of  salt  solution.  This  gives  a  dilution  of  i  to  16.  Having  mixed  as 
before,  again  withdraw  i  c.c.  of  the  mixture  and  add  it  to  the  i  c.c.  in  the 
next  tube.  We  now  have  a  dilution  of  i  to  32.  Again  withdrawing  i  c.c. 
and  adding  it  to  the  fourth  tube  containing  i  c.c.  of  salt  solution  we  have 
a  dilution  of  i  to  64.  In  tube  i  there  is  i  c.c.  of  a  dilution  of  the  serum 
of  i  to  8;  in  tube  2,  there  is  i  c.c.  of  a  dilution  of  i  to  16;  in  tube  3,  of 
i  to  32.  Tube  4  contains  2  c.c.  of  i  to  64.  Now  adding  i  c.c.  of  a  cul- 
ture of  typhoid  or  any  other  organism,  we  have  the  dilution  of  the 
serum  in  each  tube  doubled.  Tube  i  now  contains  a  serum  in  dilution 
of  i  to  16,  acting  on  the  bacteria;  tube  2  of  a  i  to  32;  tube  3  of  i  to  64. 
Now  place  these  tubes  in  the  incubator  and  after  2-5  hours  or  over- 
night, we  examine  for  the  clearing  up  of  the  supernatant  fluid.  If  the 
serum  in  a  certain  dilution  agglutinates,  the  clumps  gravitate  to  the 
bottom  and  the  upper  part  becomes  clear.  If  so  desired,  these  dilutions 
may  be  carried  on  to  i  to  several  hundred  in  the  same  way.  It  is  safer 
to  work  with  dead  cultures  instead  of  living  ones.  To  prepare,  in- 
oculate a  flask  of  bouillon  containing  about  150  c.c.  with  typhoid  or  any 
other  culture.  Allow  to  grow  for  1 8  to  24  hours  and  then  add  i  c.c.  of 
formalin.  One  percent  of  formalin  is  frequently  used  to  kill  the  cul- 
tures. At  the  end  of  24  hours  the  sterile  cultures  may  be  used  as 
with  the  live  cultures.* 

*  A  very  convenient  method  in  general  use  in  Germany  is  the  following:  Make 
dilutions  of  serum  in  ordinary  test-tubes  (J  by  6  inches)  as  described  for  the  small 


128  PRACTICAL  METHODS  IN  IMMUNITY. 

H^MOLYTIC  EXPERIMENTS. 

Take  the  blood  of  the  animal  that  has  been  used  to  immunize  the 
rabbit  and  receive  it  in  a  graduated  centrifuge  tube  containing  salt 
solution  which  has  had  i%  of  sodium  citrate  added  to  it.  This 
prevents  the  coagulation  of  the  blood.  After  mixing,  centrifuge  and 
pipette  off  supernatant  fluid.  Note  the  graduation  reached  by  the 
sediment  of  red  cells  and  make  up  with  salt  solution  to  20  times  its 
volume.  If  the  cells  reach  the  1/2  c.c.  mark,  add  10  c.c.  of  salt  solu- 
tion. This  gives  a  5%  emulsion  of  red  cells — the  percentage  usually 
used  in  hemolytic  experiments.  To  carry  out  the  test,  simply  add  i  c.c. 
of  this  5%  mixture  of  red  cells  to  each  of  the  series  of  tubes  containing 
diluted  serum,  as  with  the  macroscopic  agglutination  tests.  Place  in 
the  incubator  for  2-5  hours.  The  red  cells  settle  to  the  bottom,  and 
tubes  showing  haemolysis  have  a  light  reddish  to  rich  haemoglobin 
color.  Tubes  not  showing  haemolysis  remain  white. 

BACTERIOLYTIC  EXPERIMENTS. 

These  may  be  carried  out  in  the  peritoneal  cavity  of  a  guinea  pig, 
injecting  mixtures  of  immune  sera  and  the  bacterial  culture. 

Upon  withdrawing,  after  15-60  minutes,  the  bacteria  are  granular 
and  disintegrated.  This  is  the  well-known  Pfeiffer's  phenomenon, 
and  was  once  considered  the  most  important  test  for  cholera.  See 
Cholera.  There  have  been  several  accidents  (death  of  Orgel)  with 
this  test,  and  it  is  not  practicable  except  in  a  well  equipped  laboratory. 
Instead  of  a  guinea-pig,  we  may  simply  take  a  fresh  serum  of  known 
dilution,  and  mix  it  with  an  equal  quantity  of  the  bacterial  emulsion 
in  a  capillary  pipette;  sealing  off  the  end  of  the  pipette,  we  incubate  for 
15  minutes.  Then  filing  off  the  end  we  mix  the  culture  thoroughly  on  a 

test-tubes.  Then  take  a  loopful  (2  mg.)  of  culture  from  an  18  to  24  hour  old  agar 
culture  and  emulsify  it  thoroughly  in  the  dilution  in  the  first  test-tube — repeat  the 
process  in  the  second  tube  and  so  on.  This  procedure  is  much  oafer  than  when 
live  cultures  are  added  with  a  pipette.  Again,  the  dilution  is  unchanged  by  this 
addition  whereas  it  is  doubled  when  an  equal  volume  of  culture  is  added  to  the 
diluted  scrum.  A  control  should  always  be  made  in  normal  salt  solution.  After 
incubating,  observe  flocculent  precipitates  (agglutination)  by  tilting  the  fluid  in  the 
tubes  to  form  a  thin  layer  and  to  obtain  the  most  advantageous  light  and  look  for 
a  fine  curdy  precipitate  (agglutination)  or  a  uniformly  turbid  emulsion  (negative 
reaction) . 


COLON   BACILLUS   IN   WATER  ANALYSIS.  113 

number  of  organisms  developing  at  38°  C.  at  all  approximates  the  num- 
ber developing  at  20°  C.,  there  is  a  strong  suspicion  that  sewage  or- 
ganisms may  be  present.  Normal  waters  give  proportions  of  i  to  25 
or  i  to  50,  while  in  sewage  contaminated  waters  the  proportion  may  be 
as  i  to  4  or  less. 

In  addition,  the  appearance  of  pink  colonies  on  the  lactose  litmus 
agar  is  of  great  assistance  in  judging  of  a  water.  Both  sewage  strepto- 
cocci and  the  colon  bacillus  give  pink  colonies — those  of  the  streptococci 
are  smaller  and  more  vermilion  in  color.  Microscopic  examination 
will  differentiate  the  cocci  from  the  bacilli.  It  is  well  to  bear  in  mind 
that  the  pink  colonies  after  24  hours  may  turn  blue  in  48  hours  from 
the  development  of  ammonia  and  amines.  Consequently  the  lactose 
litmus  agar  plates  should  be  studied  after  24  hours. 

A  good  water  supply  will  rarely  show  a  pink  colony,  while  in  a 
sewage  contaminated  one  the  pink  colonies  will  probably  predominate. 

The  diagnostic  characteristics  considered  important  by  the  Ameri- 
can authorities  in  reporting  the  colon  bacillus  are: 

1.  Typical  morphology,  nonsporing  bacillus,  relatively  small  and 
often  quite  thick. 

2.  Motility  in  young  broth  cultures.  (This  is  at  times  unsatisfac- 
tory, as  some  strains  of  the  colon  bacillus  do  not  show  it  even  in  young 
bouillon  cultures.) 

3.  Gas  formula  in  dextrose  broth.     Of  about  50%  of  gas  produced, 
1/3  should  be  absorbed  by  a  2%  solution  of  sodium  hydrate  (CO2).   The 
remaining  gas  is  hydrogen.     (Later  views  indicate  that  the  gas  formula 
is  exceedingly  variable  and  should  not  be  depended  upon.     To  carry 
out  this  test  one  fills  the  bulb  of  a  fermentation  tube  with  the  caustic 
soda  solution  then,  holding  the  thumb  over  the  opening  or  with  a 
rubber  stopper,  the  bouillon  culture  and  the  soda  solution  are  mixed  by 
tilting  the  fermentation  tube  to  and  fro.     The  total  amount  of  gas  is 
first  recorded  and  then  that  remaining  after  the  CO2  has  been  absorbed 
is  reported  as  hydrogen.) 

4.  Nonliquefaction  of  gelatin. 

5.  Fermentation  of  lactose  with  gas  production. 

6.  Indol  production. 

7.  Reduction  of  nitrates  to  nitrites. 
8 


114  BACTERIOLOGY    OF    WATER,    AIR,    MILK,    ETC 

NOTE. — The  reduction  of  neutral  red  with  a  greenish-yellow 
fluorescence  is  very  striking  and  has  been  suggested  as  a  test  for  the 
colon  bacillus.  Many  other  organisms,  especially  those  of  the  hog 
cholera  group,  have  this  power.  It  is  convenient,  however,  to  color 
glucose  bouillon  with  about  i%  of  a  1/2%  solution  of  neutral  red. 

Isolation  of  the  Typhoid  Bacillus  from  Water. 

This  is  probably  the  most  discouraging  procedure  which  can  be 
taken  up  in  a  laboratory.  Only  the  most  recent  reports  of  such  isola- 
tion from  water  supplies,  which  have  been  verified  by  immunity  reac- 
tions, can  be  accepted  and  of  these  the  number  of  instances  is  exceed- 
ingly small.  Owing  to  the  long  period  of  incubation,  the  typhoid 
organisms  may  have  died  out  before  the  outbreak  of  an  epidemic 
suggests  the  examination  of  the  water  supply. 

There  have  been  various  methods  proposed  for  the  detection  of 
the  B.  typhosus  in  water.  A  method  which  would  offer  about  as 
reasonable  a  chance  of  success  as  any  other  would  be  to  pass  2  or  3 
liters  of  the  water  through  a  Berkefeld  filter;  then  to  take  up  in  a 
small  quantity  of  water  all  the  bacteria  held  back  by  the  filter.  Then 
plate  out  on  lactose  litmus  agar  and  examine  colonies  which  do  not 
show  any  pink  coloration.  The  dysentery  bacillus  has  about  the  same 
cultural  characteristics  as  the  typhoid  one,  so  that  it  is  important  to 
note  motility.  If  from  such  a  colony  you  obtain  an  organism  giving 
the  cultural  characteristics  of  B.  typhosus,  carry  out  agglutination  and 
preferably  bacteriolytic  tests  as  well.  Some  strains  of  typhoid,  espe- 
cially when  recently  isolated  from  the  body,  do  not  showr  agglutination. 

The  Conradi  Drigalski,  the  malachite-green  and  various  caffeine 
containing  plating  media  have  been  highly  recommended. 

Isolation  of  the  Cholera  Spirillum  from  Water. 

The  method  proposed  by  Koch  in  1893  does  not  seem  to  have  been 
improved  upon  by  later  investigators.  To  100  c.c.  of  the  suspected 
water  add  i%  of  peptone  and  i%  of  salt.  Incubate  at  38°  C.,  and  at 
intervals  of  8,  12  and  18  hours  examine  microscopically  loopfuls  taken 
from  the  surface  of  the  liquid  in  the  flask.  So  soon  as  comma-shape 


BACTERIOLOGICAL   EXAMINATION   OF   MILK.  115 

organisms  are  observed,  plate  out  on  agar.  The  colonies  showing 
morphologically  characteristic  organisms  should  be  tested  as  to  ag- 
glutination and  bacteriolysis.  Inasmuch  as  the  true  cholera  spirillum 
shows  a  marked  cholera-red  reaction  it  is  well  to  inoculate  a  tube  of 
peptone  solution  from  such  a  colony  and  add  a  drop  of  concentrated 
sulphuric  acid  after  incubating  for  18  hours.  The  rose-pink  colora- 
tion is  given  by  the  cholera  spirillum  with  the  acid  alone — the  nitroso 
factor  in  the  reaction  being  produced  by  the  organism. 

BACTERIOLOGICAL  EXAMINATION  OF  MILK. 

A  bacterial  milk  count  is  of  comparatively  little  value  as  showing 
whether  a  milk  is  dangerous  or  not.  As  a  matter  of  fact,  a  milk  which 
contains  several  million  of  bacteria  per  c.c.  might  be  less  dangerous 
than  one  containing  only  a  few  thousand,  especially  if  in  the  latter 
there  were  numerous  liquefiers  and  gas  producers  present.  There  is, 
however,  one  point  of  importance  in  connection  with  the  quantitative 
estimation  of  bacteria  in  milk,  and  that  is  the  fact  that  in  order  to  keep 
the  development  of  the  bacteria  within  the  limits  of  10,000  to  50,000 
per  c.c.,  it  is  necessary  that  the  requirements  of  cleanliness  in  milking 
and  the  rapid  cooling  of  the  milk  after  obtaining  it  and  the  keeping 
of  the  temperature  below  50°  C.  be  rigidly  observed.  If  a  milk  has  a 
high  count  it  shows  some  error  in  the  handling  of  the  milk.  In  making 
a  quantitative  bacteriological  examination,  the  principle  is  the  same 
as  with  water. 

Make  a  known  dilution  of  the  milk  with  sterile  water;  add  definite 
quantities  of  this  diluted  milk  to  tubes  of  melted  agar  or  gelatin  and 
pour  into  plates.  The  diluted  milk  may  also  be  delivered  in  the  center 
of  the  plate  and  the  melted  agar  or  gelatin  poured  directly  on  it, 
mixing  thoroughly.  Always  shake  the  bottle  well  before  taking 
sample. 

Example:  Added  i  c.c.  of  milk  to  199  c.c.  of  sterile  water  in  a 
large  flask  (500  to  1000  c.c.).  After  shaking  thoroughly,  take  i  c.c. 
of  this  i :  200  dilution  and  add  it  to  99  c.c.  of  sterile  water.  Shak- 
ing thoroughly,  we  have  a  dilution  of  i :  20,000.  Of  this  we  added 
.5  c.c.  to  a  tube  of  gelatin  or  agar.  After  incubation  the  plate  showed 


Il6  BACTERIOLOGY    OF    WATER,    AIR,    MILK;    ETC. 

75  colonies.  Therefore  the  milk  contained  in  each  c.c.  75  x  2  x  20,000 
(dilution)  =  3,000,000 — the  number  of  bacteria  in  each  c.c.  of  milk. 

Lactose  litmus  gelatin  or  agar  is  to  be  preferred  in  milk-work,  as 
the  normal  lactic  acid  bacteria  produce  reddish  colonies  which  are  very 
striking.  A  standard  easily  attained  for  high-grade,  certified  milk 
would  be  5,000  to  10,000  per  c.c. 

In  the  qualitative  examination  of  milk,  many  dairies  employ  the 
fermentation  tube,  any  organism  producing  gas  being  considered 
undesirable.  Again  liquefying  organisms,  as  shown  by  the  presence 
of  such  bacteria  in  the  gelatin  plates,  is  evidence  of  probable  contami- 
nation by  fecal  bacteria.  A  question  which  seems  difficult  to  decide 
is  as  to  the  general  nature  of  the  so-called  normal  lactic  acid  bacteria 
of  milk.  Some  describe  them  as  very  short,  broad  bacilli  with  very 
small  colonies,  fermenting  lactose  with  the  formation  of  lactic  acid. 
Others  consider  that  the  streptococci  are  the  organisms  which  are 
concerned  with  the  normal  fermentative  changes.  In  examining 
specimens  of  milk  considered  the  best  on  the  market,  I  have  repeatedly 
found  the  small  red  colonies  on  lactose  litmus  agar  to  be  streptococci. 
In  connection  with  the  organisms  present  in  the  tablets  used  for  treat- 
ing milk  to  produce  lactic  acid  for  the  treatment  of  intestinal  disorders, 
and  considered  to  be  normal  lactic  acid  bacteria,  I  have  found  both 
streptococci  and  bacilli.  These  have  all  agreed,  however,  in  not  pro- 
ducing gas  in  either  lactose  or  glucose  fermentation  tubes. 

Another  source  of  information  as  to  the  quality  of  a  milk  may  be 
derived  from  a  study  of  the  number  of  leukocytes  or  pus  cells  con- 
tained in  i  c.c.  of  the  milk. 

The  Doane-Buckley  method  is  probably  the  most  accurate.  In 
this  you  throw  down  the  cellular  contents  of  10  c.c.  of  milk  in  a  cen- 
trifuge revolving  about  1000  times  a  minute  for  ten  to  twenty  minutes. 
Then  remove  supernatant  milk  and  add  0.5  c.c.  of  Toisson's  solution 
to  the  sediment.  You  thus  have  the  leukocytes  of  10  c.c.  contained  in 
0.5  c.c.  (Concentrated  twenty  times.)  Make  a  haemacytometer  pre- 
paration as  for  blood  and  find  the  average  number  of  cells  for  each 
square  millimeter.  Then  multiply  this  by  10  to  get  the  number  of 
cells  in  a  cubic  millimeter.  As  a  cubic  millimeter  is  one  thousand 
times  smaller  than  a  cubic  centimeter,  you  multiply  the  number  per 


BACTERIOLOGICAL   EXAMINATION   OF  AIR.  117 

cubic  millimeter  by  one  thousand.  Then,  as  the  milk  was  concentrated 
twenty  times,  you  divide  by  20.  (If  it  were  diluted  twenty  times, 
you  would  multiply  by  20.) 

Example:     Found  an  average  of  50  cells  per  square  millimeter. 
This  would  make  500  per  cubic  millimeter,  and  500,000  per  c.c.; 
.  then  500,000  divided  by  20  would  give  25,000. 

|There  is  no  agreement  as  to  a  standard  for  allowable  leukocytes. 
Even  in  apparently  healthy  animals  they  may  exceed  100,000  per  c.c. 
Doane  has  suggested  500,000  per  c.c.  as  a  preferable  limit. 

The  smear  methods  for  determining  the  number  of  leukocytes 
present  do  not  compare  in  accuracy  with  the  volumetric  ones. 

BACTERIOLOGICAL  EXAMINATION  OF  AIR. 

In  Paris  a  cubic  meter  of.  air  was  found  to  contain  the  following 
number  of  organisms: 

Suburbs. — Winter,       145  moulds,  170  bacteria. 

Summer,      245  moulds,  345  bacteria. 

City  Hall. — Winter,     1345  moulds,  4305  bacteria. 

Summer,  2500  moulds,  9845  bacteria. 

Air  of  hospitals,  especially  after  sweeping,  may  contain  50,000 
bacteria  per  cubic  meter.  There  does  not  seem  to  be  any  particular 
relation  between  the  amount  of  carbon  dioxide  in  air  and  the  bacterial 
content. 

Petri's  Rough  Method.— Exposure  of  a  lactose  litmus agar  plate 
(capacity  100  sq.  cm.)  for  five  minutes  will  give  the  number  of  organ- 
isms present  in  ten  liters  of  air.  Multiply  by  100  for  one  cubic  meter. 

The  two  groups  of  organisms  usually  found  in  air  are  (i)  bacteria 
and  (2)  moulds.  Moulds  (spores)  may  be  carried  by  currents  of  air; 
bacteria,  however,  are  generally  carried  about  by  particles  of  dust  or 
finely  divided  liquids  (spray).  On  the  lactose  litmus  agar  plate 
staphylococci  and  streptococci  show  as  bright  red  colonies. 

Sedgwick  Tucker  Sterile  Granulated  Sugar  Method. — Sterilize 
aerobioscope  and  introduce  granulated  sugar  on  support.  Again 
sterilize  (not  over  120°  C.  in  dry-air  sterilizer).  Allow  a  given  quan- 
tity of  air  to  pass  through;  then  shake  the  sugar  into  wide  part  of 


Il8  BACTERIOLOGY    OF    WATER,    AIR,    MILK,    ETC. 

aerobioscope.  Now  pour  in  10  or  15  c.c.  of  melted  gelatin  (40°  C.)  to 
dissolve  sugar.  Roll  tubes  as  for  Esmarch  roll  cultures,  and  incubate 
at  room  temperature.  To  draw  air  through  the  aerobioscope,  connect 
the  small  end  with  a  piece  of  rubber  tubing  which  is  attached  to  a  tube 
in  the  stopper  of  an  aspirating  bottle.  Having  poured  a  definite 
quantity  of  water  into  the  aspirating  bottle,  allow  the  water  to  run  out.. 
The  same  quantity  of  air  will  be  drawn  through  the  sugar  of  the 
aerobioscope  as  the  amount  of  water  passing  out  of  the  aspirating 
bottle.  The  bacteria  and  moulds  are  caught  by  the  sugar. 

Example. — Passed  10  liters  of  air  through  the  aerobioscope.     The 
bacteria  in  this  quantity  of  air  showed  75  colonies  when  incubated 


FIG.  43. — Sedgwick-Tucker  aerobioscope.     (Williams.) 

at  20°  C.  The  unit  being  one  cubic  meter  or  one  thousand  liters, 
we  have  only  obtained  the  bacteria  of  one  hundredth  of  the  unit. 
Hence  multiplying  75  by  100  gives  7,500  bacteria  as  present  in  one 
cubic  meter  of  the  air  examined. 

In  comparing  the  results  with  the  aerobioscope  with  those  obtained 
by  exposing  a  plate  as  in  Petri's  method  for  ten  instead  of  five  minutes, 
it  was  found  that  the  latter  was  sufficiently  in  accord  to  make  it  a 
satisfactory  approximate  quantitative  method.  The  simplicity  and 
ease  of  access  of  the  colonies  developing  in  it  make  it  preferable  when 
the  air  of  operating-rooms  or  hospital  wards  is  to  be  examined. 


CHAPTER  XII. 
PRACTICAL  METHODS  IN  IMMUNITY. 

THAT  which  prevents  the  gaining  of  a  foothold  by  disease  organisms 
in  the  animal  body  or  which  neutralizes  their  harmful  products  or 
destroys  the  parasites  is  termed  immunity.  In  the  main,  the  question 
of  immunity  hinges  on  the  powers  of  resistance  of  the  human  body 
and  the  aggressiveness  or  virulence  of  the  invading  organism.  It 
must  always  be  kept  in  mind  that  immunity  is  only  relative;  thus  the 
fowl,  which  is  practically  immune  to  tetanus,  may  be  made  to  suc- 
cumb by  reducing  its  resistance  by  refrigeration  or  by  increasing  the 
amount  of  poison  introduced.  The  insusceptibility  which  the  fowl 
has  to  tetanus  or  which  man  has  to  many  diseases  of  animals  is  best 
termed  inherent  immunity,  and  is  at  present  only  a  subject  of  theo- 
retical interest.  When  immunity  to  a  given  disease  is  obtained  as  a 
result  of  an  attack  of  the  disease  in  question  or  by  laboratory  methods 
of  inoculation,  this  is  termed  properly  an  acquired  immunity,  and  in 
the  former  case  is  a  naturally  acquired  immunity  or  " natural  im- 
munity" and  in  the  second  is  an  artificially  acquired  immunity  or 
" artificial  immunity." 

As  a  result  of  an  attack  of  a  disease  or  in  response  to  the  stimulus 
of  the  injection  of  the  organism  or  its  products,  we  have  developed  in 
the  man  so  injected  certain  specific  antagonistic  properties  to  that 
organism,  which  are  usually  demonstrable  in  the  blood  serum  or 
other  body  fluids,  and  to  which  we  apply  the  terms  agglutinating 
power,  opsonic  power  or  bacteriolytic  power.  The  term  antibody  is 
also  applied.  All  three  powers  may  be  present  together  in  equal  or  in 
varying  degree  or  one  or  more  may  be  absent.  By  agglutinating 
power  we  mean  that  which  causes  evenly  distributed  organisms  to 
come  together  and  form  clumps.  By  opsonic  power  we  mean  that 
which  so  alters  the  resistance  of  bacteria  that  the  phagocytes  ingest 
them.  By  bacteriolytic  power  we  mean  that  which  brings  about 

119 


120 


PRACTICAL    METHODS    IN    IMMUNITY. 


disintegration  or  lysis  of  the  specific  organism.  The  bacterium 
which  causes  the  disease  or  which  is  used  in  inoculation  for  the  pro- 
duction of  immunity  is  termed  the  specific  organism. 

Of  the  different  kinds  of  immunity  only  artificial  immunity  will 
be  considered.  This  may  be  obtained  in  two  ways :  i .  By  injecting  the 
bacteria  or  their  products  into  man  or  animals  and  as  the  result  of 
the  activity  of  the  cells  of  the  animal  invaded,  antibodies  are  formed 

which  neutralize  the  toxins  of 
or  destroy  the  specific  bac- 
teria. These  antibodies  which 
are  supposed  to  be  thrown  off 
(free  receptors)  or  which  may 
remain  attached  to  the  cell 
(sessile  receptors)  may  re- 
main potential  for  months  or 
years  and  so  confer  a  more 
or  less  enduring  immunity. 
This  is  termed  active  im- 
munity. 2.  When  we  take 
the  serum  of  a  man  or  animal 
immunized  actively  and  inject 
it  with  its  contained  anti- 
bodies into  a  second  animal 
or  man,  we  confer  an  im- 
munity on  the  second  animal; 
but  as  his  cells  take  no  active 
part  in  the  production  of  the 
immunity,  but  are  only  pas- 
sive, we  term  this  immunity  "passive  immunity."  If  this  serum  which 
is  introduced  in  passive  immunity  only  neutralizes  the  toxic  products 
of  the  infecting  bacteria,  we  term  it  antitoxic  passive  immunity  and 
designate  the  immune  serum  as  antitoxic  serum.  If  it  destroys  the 
organism,  we  call  it  antimicrobic  serum,  and  the  immunity,  antimi- 
crobic  passive  immunity.  Some  immune  sera  are  both  antitoxic  and 
antimicrobic. 

It  is  well  to  remember  that  some  organisms  produce  a  toxin  which 


FIG.  44. — Receptors  of  the  first  order  uniting 
with  toxin.  (Journal  of  the  American  Medi- 
cal Association.  1905.  P.  955.) 

a,  Cell  receptor;  6;  toxin  molecule;  c,  hap- 
tophore  of  the  toxin  molecule;  d,  toxophore 
of  the  toxin  molecule;  e,  haptophore  of  the 
cell  receptor. 


ANTITOXIC  AND  ANTIMICROBIC   SERA. 


121 


is  given  off  while  the  bacterium  is  alive;  and  in  other  instances  the  toxin 
is  intracellular  and  is  oily  given  off  when  the  bacterium  disintegrates; 
consequently,  an  antimicrobic  serum  may  cause  the  liberation  of  toxin. 
Diphtheria,  tetanus  or  botulism  antisera  are  instances  of  antitoxic 
sera,  while  practically  all  others  are  antimicrobic.  There  is  but  one 
factor  to  consider  in  an  antitoxic  serum  and  that  is  the  protoplasmic 
particles  which  are  thrown 
off  from  the  cell  in  response 
to  the  injury  incident  to  the 
attack  upon  the  cell  by  the 
toxin  particles.  This  free 
particle  in  the  circulation 
represents  the  entire 
mechanism  of  antitoxic  im- 
munity. It  is  capable  of 
uniting  with  the  toxin  mole- 
cule and  neutralizing  ifs 
toxic  power,  or  rather  so 
binding  its  combining  end 
(haptophore  group)  that  it 
is  incapable  of  attaching 
itself  to  a  cell,  so  that  the 
poisonous  end  of  the  toxin 
(toxophore  group)  cannot 
have  access  to  the  cell.  In 
antimicrobic  sera  we  have 
two  factors  to  consider,  the 
first  is  a  protoplasmic  par- 
ticle quite  similar  to  the  anti- 
toxin molecule,  but  which  in  itself  has  no  power  of  injuring  its  specific 
bacterium.  This  particle  is  generally  referred  to  as  the  amboceptor 
or  immune  body.  It  is  the  specific  product  of  the  activity  of  a  specific 
bacterium  or  foreign  cell  against  the  body  cells  attacked.  It  with- 
stands a  temperature  above  56°  C.  and  of  itself  is  incapable  of  injuring 
the  bacterium  in  response  to  whose  attack  it  was  produced.  The 
second  factor  in  the  bacteriolysis  of  the  specific  bacterium,  or  the 


FIG.  45. — Receptors  of  the  second  order  and 
of  some  substance  uniting  with  one  of  them. 
(Journal  oj  the  American  Medical  Association. 
1905.  P.  1113.) 

c,  Cell  recepto;  of  the  second  order;  d,  tox- 
ophore or  zymophore  group  of  the  receptor; 
e,  haptophore  of  the  receptor;  /,  Food  substance 
or  product  of  bacterial  disintegration  uniting 
with  the  haptophore  of  the  cell  receptor. 


122 


PRACTICAL   METHODS    IN    IMMUNITY. 


haemolysis  of  the  specific  foreign  cell,  is  something  normally  present 
in  the  serum  of  every  animal,  and  which  is  capable  of  disintegrating  a 
foreign  cell  or  bacterium,  provided  it  can  have  access  to  the  cell  or 
bacterium  through  an  intermediary  amboceptor  (hence  the  ambo- 
ceptor  is  sometimes  called  an  intermediary  body).  This  something 
is  called  the  "complement."  It  is  by  some  called  "alexine,"  by 
others  cytase  (MetchnikorT).  The  complement  cannot  act  upon  and 
destroy  an  invading  bacterium  or  cell  unless  the  amboceptor  is 


FIG.  46. — Receptor  of  the  third  order,  and  of  some  substance  uniting  with  one 
of  them.  (Journal  0}  the  American  Medical  Association.  1905.  P  1369  ) 

c,  Cell  receptor  of  the  third  order — an  amboceptor;  e,  one  of  the  haptophores  of 
the  amboceptor,  with  which  some  food  substance  or  product  of  bacterial  disintegra- 
tion (/)  may  unite;  g;  the  other  haptophore  of  the  amboceptor  with  which  com- 
plement may  unite;  k,  complement ;  h,  the  haptophore;  z,  the  zymotoxic  group  of 
complements. 


present  to  make  the  necessary  connection.  The  complement  is 
destroyed  by  a  temperature  of  56°  C.,  so  that,  if  we  heat  the  serum 
from  an  immune  animal  to  56°  C.,  the  complement  it  naturally  con- 
tains is  destroyed,  and  the  amboceptor  it  contains,  which  is  not 
injured  by  such  a  temperature,  is  incapable  of  destroying  bacteria  or 
cells,  unless  we  replace  the  complement  which  has  been  destroyed  by 
fresh  complement.  This  is  done  experimentally  by  adding  the  serum 


ACTIVATION   OF   IMMUNE   SERA. 


I23 


of  a  non immunized  animal  which  contains  the  complement,  but  no 
specific  immune  body  (amboceptor)  to  the  heated  serum.  This  is 
termed  "activating,"  and  a  serum  so  treated  is  said  to  be  "activated." 
When  an  immune  serum  has  been  heated  to  56°  C.,  it  is  said  to  have 
been  "inactivated". 


FIG.  47. — i,  Red  cells  +  normal  serum.  No  amboceptor.  Nohemolysis.  A.  com- 
plement; B,  normal  red  eel!.  2.  Red  cells  +  immune  serum.  Complement  and 
amboceptor.  Hemolysis.  C,  complement;  D,  amboceptor;  E,  hemolyzed  red  cell. 

3.  Red  cells  +  immune  serum  heated  to  56°  C.     Inactivated.     Complement  de- 
stroyed.    No  hemolysis.     F,  destroyed  complement;  G,  amboceptor;  H,  red  cells. 

4.  Red    cells  +  heated  immune  serum  +  fresh  serum.     (Activated  by  contained 
complement).    Hemolysis.     I,  destroyed  complement;  J,  fresh  complement;  K,  am- 
boceptor;   L,  hemolyzed   red   cell.     5,    Diagram   showing   antitoxin   production. 
a,  toxin  molecule;  6,  antitoxin  molecule;  c,  neutralization  of  toxin  by  antitoxin. 
6.  Diagram  showing  bacteriolysin.     d,  complement;  e,  amboceptor;  /,  bacillus. 


When  we  allow  a  mixture  of  bacteria  or  cells  to  remain  in  contact 
with  their  specific  immune  serum  which  has  been  inactivated,  the 
amboceptors  attach  themselves  to  the  bacteria  or  cells,  so  that  now,  upon 
adding  normal  serum  (complement),  these  bacteria  or  cells  are  so  pre- 


124  PRACTICAL    METHODS    IN    IMMUNITY. 

pared  that  the  complement  can  disintegrate  them.     This  experiment 
is  termed  "sensitizing"  and  cells  so  treated  are  said  to  be  "sensitized." 

METHODS  FOR  OBTAINING  IMMUNE  SERA. 

While  a  convalescent  from  a  disease  may  be  utilized  to  obtain  an 
antitoxic,  agglutinating,  opsonic  or  bacteriolytic  serum  against  the 
specific  bacterium,  yet  this  is  more  conveniently  obtained  from  an 
animal  which  has  been  immunized  against  the  bacterium  or  cell  in 
question.  The  rabbit  is  the  most  convenient  animal  to  employ  for  the 
production  of  immune  sera  where  the  object  is  to  have  at  hand  a  serum 
for  use  in  diagnosis. 

Where  sera  are  used  on  an  extensive  scale,  as  in  the  production  of 
curative  sera,  larger  animals  are  employed.  There  are  two  application 
of  serum  diagnosis:  i.  Where  the  bacterium  is  known  and  the  serum 
is  to  be  diagnosed.  2.  Where  the  serum  is  known  and  the  bacterium 
is  to  be  diagnosed. 

The  first  is  employed  by  testing  the  agglutinating  or  bacteriolytic 
power  of  the  serum  taken  from  a  patient  upon  pure  cultures  of  the  organ- 
ism which  is  suspected  as  the  cause  of  the  disease.  The  Widal  test  (ag- 
glutination) is  the  best  instance  of  this  procedure.  This  method  is 
of  practical  value  in  the  diagnosis  only  of  typhoid,  Malta  fever  and 
paratyphoid.  In  diseases  like  cholera  and  bacillary  dysentery,  the 
disease  has  run  its  course  before  agglutinating  power  becomes  apparent 
in  the  serum.  This  method,  however,  may  be  used  to  prove  that 
a  convalescent  has  suffered  from  a  suspected  disease.  Thus,  by  test- 
ing the  agglutinating  power  of  a  serum,  one  or  two  weeks  after  re- 
covery from  a  suspicious  case  of  ptomaine  poisoning,  we  may  be  able 
to  demonstrate  that  the  case  in  question  was  cholera.  The  second 
method  has  wider  application,  and  is  the  one  in  which  we  use  the  sera 
of  animals  which  have  been  immunized  with  known  bacteria.  Or- 
ganisms isolated  from  urine,  faeces  or  blood  of  patients,  or  those  obtained 
from  water  or  food  supplies  may  be  identified  by  testing  the  agglu- 
tinating, opsonic  or  bacteriolytic  power  of  known  sera  against  them. 
This  has  a  wide  range  of  applicability.  The  testing  of  the  opsonic 
power  of  the  sera  in  man  or  animals  immunized  against  plague,  and 
possibly  cerebrospinal  meningitis,  seems  to  give  more  definite  informa- 


METHODS    FOR   OBTAINING   IMMUNE    SERA.  125 

tion  than  do  agglutination  or  bacteriolytic  tests.  With  the  majority 
of  other  organisms,  however,  the  agglutination  test  is  the  one  almost 
always  preferred. 

Even  in  a  small  laboratory  there  are  no  particular  difficulties  in  the 
way  of  having  on  hand  rabbits  immunized  against  typhoid,  paratyphoid 
Malta  fever,  acid  producing  and  nonacid  producing  strains  of  dysentery, 
cholera,  etc.  Just  as  we  inject  men  with  vaccines  prepared  from 
various  bacteria  in  opsonic  therapy,  so  we  inject  animals  to  produce  sera 
for  diagnosis.  We  may  use  either  a  bouillon  culture  or  the  growth  on 
agar.  slants  taken  up  with  salt  solution  as  the  inoculating  material. 
This  is  heated  for  one  hour  at  60°  C.  to  kill  the  bacteria.  Where  we 
desire  to  produce  a  serum  which  will  disintegrate  red  blood  cells 
(haemolytic  serum),  we  inject  about  4  c.c.  of  the  defibrinated  blood  of 
the  animal  for  which  we  wish  to  produce  a  specific  serum.  Thus  for 
a  serum  for  use  in  a  medicolegal  case  we  would  inject  the  rabbit  with 
human  blood.  The  most  convenient  way  to  defibrinate  blood  is  to 
break  a  section  of  glass  tubing  into  fragments,  put  these  fragments 
into  a  glass  test-tube,  sterilize  tube  and  contents  in  a  flame  or  sterilizer 
and,  when  cool,  let  the  blood  drop  into  the  test-tube  (we  may  use  a 
Wright's  pipette  with  a  rubber  bulb  to  take  up  the  blood  from  a  punc- 
ture of  the  finger  and  eject  it  into  the  tube).  By  shaking,  the  fibrin 
collects  on  the  glass  fragments,  and  we  have  the  corpuscular  emulsion 
to  inject.  Inject  about  4  c.c.  of  the  defibrinated  blood  or  i  c.c.  of  the 
killed  bacterial  bouillon  culture  into  the  peritoneal  cavity  of  the  rabbit. 
The  easiest  wray  to  inject  the  rabbit  is  to  hold  the  animal  head  down 
and  plunge  the  needle  in  the  median  line  into  the  abdominal  cavity, 
forcing  in  the  contents  of  the  syringe.  The  intestines  gravitate  down- 
ward and  by  entering  the  needle  below  the  limits  of  the  bladder 
we  avoid  injuring  any  vital  part.  It  may  be  more  satisfactory  to  at 
first  inject  only  about  1/2  c.c.,  and  then  if  there  is  very  little  reaction, 
as  shown  by  the  appetite  and  spirits  of  the  rabbit,  to  inject  about  4 
days  later  i  c.c.  About  4  or  5  injections  at  intervals  of  3  to  5  days 
will  usually  produce  an  immune  serum.  Some  animals  do  not  seem 
to  be  capable  of  producing  antibodies,  so  that  it  may  be  necessary  to 
use  one  or  more  rabbits  before  a  satisfactory  serum  is  obtained.  The 
most  convenient  way  of  obtaining  serum  for  a  test  is  to  cut  across  one 


126  PRACTICAL    METHODS    IN    IMMUNITY. 

of  the  marginal  veins  of  the  rabbit's  ear,  and  collect  the  blood  in  a 
Wright's  U-tube.  Centrifugalizing,  we  have  the  serum  ready  for  use. 
The  immune  body  and  agglutinin  in  serum  remain  active  for  weeks 
when  kept  in  the  refrigerator.  The  complement  and  opsonin,  however, 
begin  to  deteriorate  at  once  and  have  disappeared  by  the  fifth  day. 
Consequently,  for  opsonic  and  bacteriolytic  and  haemolytic  experiments, 
fresh  serum — 12  to  24  hours — must  be  used,  or  it  may  be  activated. 

AGGLUTINATION  TESTS. 

There  are  two  methods  of  testing  the  agglutinating  powers  of  a 
serum — the  microscopical  and  the  macroscopical  or  sedimentation 
method. 

i.  For  the  microscopical  method  draw  up  serum  to  the  mark  .5 
of  the  white  pipette.  Then  draw  up  salt  solution  to  the  mark  n. 
This  when  mixed  gives  a  dilution  of  i  to  20.  One  loopful  of  the  diluted 
serum  and  one  loopful  of  a  bouillon  culture  or  salt  solution  suspen- 
sion of  the  organism  to  be  tested  gives  a  dilution  of  i  to  40.  One  loop- 
ful of  the  1-20  diluted  serum  and  3  loopfuls  of  the  bacterial  suspension 
give  a  dilution  of  1-80.  These  two  dilutions  answer  in  ordinary  diag- 
nostic tests.  The  red  pipette  with  a  i-ioo  or  1-200  dilution  may  be 
used  where  dilutions  approaching  i-iooo  are  desired.  Having  mixed 
the  diluted  serum  and  the  bacterial  suspension  on  a  cover-glass,  we 
invert  it  over  a  vaselined  concave  slide  and  examine  with  a  high 
power,  a  dry  objective  (1/6  in.).  It  is  simpler  to  make  a  ring  of  vaselin 
to  fit  the  cover-glass  and  make  the  mixture  of  diluted  serum  and  culture 
in  the  center  of  this  ring  or  square.  Then  apply  the  cover-glass,  press 
it  down  on  the  vaselin  ring  and  examine  as  with  the  ordinary  hanging 
drop.  In  making  dilutions  it  is  preferable  to  use  salt  solution,  as  the 
phenomenon  of  agglutination  requires  the  presence  of  salts.  Ordi- 
narily, 30  minutes  is  a  sufficient  time  to  wait  before  reporting  the 
absence  of  agglutination.  Agglutination  is  more  rapid  at  body  tempera- 
ture than  at  room  temperature.  In  reporting  agglutination,  always 
give  time  and  dilution.  It  is  absolutely  necessary  that  a  control  prepa- 
ration be  prepared  in  every  instance;  that  is,  one  with  the  bacterial 
culture  alone  or  with  a  normal  serum  of  the  same  dilution  as  the  lowest 
used.  Some  normal  sera  will  agglutinate  in  i  to  10  dilution,  and  group 


MACROSCOPICAL  AGGLUTINATION.  127 

agglutinations  (as  paratyphoid  with  typhoid  serum)  may  occur  in  i 
to  40  or  possibly  higher.  It  is  very  unusual  for  sera  to  agglutinate 
any  other  bacteria  than  its  specific  one  in  dilutions  as  high  as  1-80. 
2.  For  the  macroscopical  or  sedimentation  test,  take  a  series  of 
small  test-tubes  (3/8  x  3  in.)  and  deposit  i  c.c.  of  salt  solution  in  each 
of  the  series.  Now,  having  taken  an  empty  test-tube,  drop  4  drops  of 
serum  in  it  and  then  add  1 2  drops  of  salt  solution.  This  approximately 
gives  i  c.c.  of  a  1-4  dilution  of  the  serum.  With  a  rubber-bulb  capillary 
pipette,  which  has  been  graduated  to  hold  16  drops  or  i  c.c.  draw  up  the 
contents  of  the  tube  containing  the  i  to  4  serum  and  add  it  to  the  next 
tube  containing  i  c.c.  of  salt  solution.  This  gives  a  dilution  of  i  to  8. 
Now  mix  thoroughly  by  drawing  up  and  forcing  out  with  the  bulb 
pipette,  and  then  withdraw  i  c.c  and  add  to  the  next  tube  containing 
i  c.c.  of  salt  solution.  This  gives  a  dilution  of  i  to  16.  Having  mixed  as 
before,  again  withdraw  i  c.c.  of  the  mixture  and  add  it  to  the  i  c.c.  in  the 
next  tube.  We  now  have  a  dilution  of  i  to  32.  Again  withdrawing  i  c.c. 
and  adding  it  to  the  fourth  tube  containing  i  c.c.  of  salt  solution  we  have 
a  dilution  of  i  to  64.  In  tube  i  there  is  i  c.c.  of  a  dilution  of  the  serum 
of  i  to  8;  in  tube  2,  there  is  i  c.c.  of  a  dilution  of  i  to  16;  in  tube  3,  of 
i  to  32.  Tube  4  contains  2  c.c.  of  i  to  64.  Now  adding  i  c.c.  of  a  cul- 
ture of  typhoid  or  any  other  organism,  we  have  the  dilution  of  the 
serum  in  each  tube  doubled.  Tube  i  now  contains  a  serum  in  dilution 
of  i  to  1 6,  acting  on  the  bacteria;  tube  2  of  a  i  to  32;  tube  3  of  i  to  64. 
Now  place  these  tubes  in  the  incubator  and  after  2-5  hours  or  over- 
night, we  examine  for  the  clearing  up  of  the  supernatant  fluid.  If  the 
serum  in  a  certain  dilution  agglutinates,  the  clumps  gravitate  to  the 
bottom  and  the  upper  part  becomes  clear.  If  so  desired,  these  dilutions 
may  be  carried  on  to  i  to  several  hundred  in  the  same  way.  It  is  safer 
to  work  with  dead  cultures  instead  of  living  ones.  To  prepare,  in- 
oculate a  flask  of  bouillon  containing  about  150  c.c.  writh  typhoid  or  any 
other  culture.  Allow  to  grow  for  18  to  24  hours  and  then  add  i  c.c.  of 
formalin.  One  percent  of  formalin  is  frequently  used  to  kill  the  cul- 
tures. At  the  end  of  24  hours  the  sterile  cultures  may  be  used  as 
with  the  live  cultures.* 

*A  very  convenient  method  in  general  use  in  Germany  is  the  following:   Make 
dilutions  of  serum  in  ordinary  test-tubes  (J  by  6  inches)  as  described  for  the  small 


128  PRACTICAL  METHODS  IN  IMMUNITY. 

H^MOLYTIC  EXPERIMENTS. 

Take  the  blood  of  the  animal  that  has  been  used  to  immunize  the 
rabbit  and  receive  it  in  a  graduated  centrifuge  tube  containing  salt 
solution  which  has  had  i%  of  sodium  citrate  added  to  it.  This 
prevents  the  coagulation  of  the  blood.  After  mixing,  centrifuge  and 
pipette  off  supernatant  fluid.  Note  the  graduation  reached  by  the 
sediment  of  red  cells  and  make  up  with  salt  solution  to  20  times  its 
volume.  If  the  cells  reach  the  1/2  c.c.  mark,  add  10  c.c.  of  salt  solu- 
tion. This  gives  a  5%  emulsion  of  red  cells — the  percentage  usually 
used  in  hemolytic  experiments.  To  carry  out  the  test,  simply  add  i  c.c. 
of  this  5%  mixture  of  red  cells  to  each  of  the  series  of  tubes  containing 
diluted  serum,  as  with  the  macroscopic  agglutination  tests.  Place  in 
the  incubator  for  2-5  hours.  The  red  cells  settle  to  the  bottom,  and 
tubes  showing  haemolysis  have  a  light  reddish  to  rich  haemoglobin 
color.  Tubes  not  showing  haemolysis  remain  white. 

BACTERIOLYTIC  EXPERIMENTS. 

These  may  be  carried  out  in  the  peritoneal  cavity  of  a  guinea-pig, 
injecting  mixtures  of  immune  sera  and  the  bacterial  culture. 

Upon  withdrawing,  after  15-60  minutes,  the  bacteria  are  granular 
and  disintegrated.  This  is  the  well-known  Pfeiffer's  phenomenon, 
and  was  once  considered  the  most  important  test  for  cholera.  See 
Cholera.  There  have  been  several  accidents  (death  of  Orgel)  with 
this  test,  and  it  is  not  practicable  except  in  a  well- equipped  laboratory. 
Instead  of  a  guinea-pig,  we  may  simply  take  a  fresh  serum  of  known 
dilution,  and  mix  it  with  an  equal  quantity  of  the  bacterial  emulsion 
in  a  capillary  pipette;  sealing  off  the  end  of  the  pipette,  we  incubate  for 
15  minutes.  Then  filing  off  the  end  we  mix  the  culture  thoroughly  on  a 

test-tubes.  Then  take  a  loopful  (2  mg.)  of  culture  from  an  18  to  24  hour  old  agar 
culture  and  emulsify  it  thoroughly  in  the  dilution  in  the  first  test-tube — repeat  the 
process  in  the  second  tube  and  so  on.  This  procedure  is  much  bafer  than  when 
live  cultures  are  added  with  a  pipelte.  Again,  the  dilution  is  unchanged  by  this 
addition  whereas  it  is  doubled  when  an  equal  volume  of  culture  is  added  to  the 
diluted  serum.  A  control  should  always  be  made  in  normal  salt  solution.  After 
incubating,  observe  flocculent  precipitates  (agglutination)  by  tilting  the  fluid  in  the 
tubes  to  form  a  thin  layer  and  to  obtain  the  most  advantageous  light  and  look  for 
a  fine  curdy  precipitate  (agglutination)  or  a  uniformly  turbid  emulsion  (negative 
reaction). 


IMMUNITY   EXPERIMENTS.  I2Q 

sterile  slide;  deposit  a  small  drop  fora  hanging-drop  preparation  and 
draw  up  the  remaining  mixture  into  the  same  tube  and  again  incubate. 
The  time  and  dilution  with  which  the  culture  becomes  nonmotile  and 
granular  (bacteriolytic  disintegration)  should  be  recorded.  Controls 
with  normal  serum  are  always  necessary. 

DEVIATION  OF  THE  COMPLEMENT. 

It  has  been  found  that  if  there  is  not  sufficient  immune  body  in  a 
mixture  of  normal  serum,  containing  abundant  complement,  and 
bacterial  emulsion,  only  a  portion  of  the  bacteria  present  will  be 
destroyed.  Increasing  the  amount  of  immune  body  with  a  constant 
quantity  of  normal  serum,  we  reach  a  point  where  all  the  bacteria  are 
destroyed.  Now,  if  we  continue  to  increase  beyond  this  point  the 
addition  of  immune  serum,  the  destruction  of  the  bacteria  ceases,  and 
the  cultures  will  again  contain  myriads  of  living  bacteria. 

To  carry  out  the  test,  make  a  series  of  tubes  containing  mixtures  of 
bacteria  with  the  same  quantity  in  each  of  normal  serum.  Thus,  each 
tube  contains  1/2  c.c.  of  bacterial  emulsion  and  1/2  c.c.  of  i-io  normal 
serum.  Now  inactivate  a  tube  of  i-ioo  immune  serum  and  to  each  of 
the  tubes  of  normal  serum  and  bacterial  emulsion  add  increasing 
drops  of  the  inactivated  i-ioo  immune  serum.  Thus,  i  drop  to  No.  i 
tube;  2  drops  to  No.  2  tube  and  so  on.  After  incubating  for  2  hours,  we 
take  a  pipette  and  plate  out  a  fraction  of  a  drop  in  an  agar  plate.  The 
limit  at  which  bacteriolysis  is  complete  is  showrn  by  there  being  an 
absence  of  colonies. 

Beyond  or  below  that  point  colonies  are  more  or  less  abundant. 
The  explanation  of  this  phenomenon  of  deviation  or  deflection  of  the 
complement  is  that  where  we  have  an  excess  of  amboceptors  for 
available  receptors  on  the  bacterial  cells,  only  a  portion  of  the  ambo- 
ceptors can  attach  themselves  to  their  specific  bacteria.  The  free 
amboceptors,  not  being  able  to  form  a  union  with  the  bacterial  cell 
receptors  (for  which  they  have  a  greater  affinity),  combine  with  the 
complement  present.  Unless  the  complement  be  in  excess,  there  will 
be  no  free  complement  left  to  join  onto  the  amboceptors  attached  to  the 
bacterial  cells,  and  consequently  bacteriolysis  does  not  take  place  and 
the  plate  cultures  show  an  abundance  of  colonies. 
9 


130  PRACTICAL  METHODS  IN  IMMUNITY. 

FIXATION  OR  ABSORPTION  OF  THE  COMPLEMENT. 

One  of  the  controversies  in  connection  with  the  nature  of  the 
complement  is  that  regarding  the  question  of  the  unity  of  complements 
or  whether  there  exist  different  kinds  of  complements  for  different 
amboceptors  (unity  and  multiplicity  of  complement).  To  prove  that 
a  single  complement  will  act  with  varying  amboceptors,  Bordet  and 
Gengou  showed  that  the  same  complement  would  activate  both 
haemolytic  and  bacteriolytic  immune  bodies.  If  to  a  mixture  of 
typhoid  bacteria  and  inactivated  typhoid  immune  serum  some 
guinea  pig  serum  is  added  and  the  mixture  be  allowed  to  remain  at 
37°  C.  for  2  hours,  and  then  sensitized  red  cells  be  added  and  the  mix- 
ture again  be  placed  in  the  incubator  for  2  hours,  no  haemolysis  will  be 
found  to  have  occurred,  because  the  bacteria  have  absorbed  all  the 
guinea-pig  complement  through  the  intervening  typhoid  amboceptors, 
and  there  is  no  complement  left  to  haemolyze  the  red  cells  through  the 
specific  blood-cell  amboceptors.  If,  instead  of  immune  typhoid  serum, 
the  serum  of  a  normal  person  had  been  used,  there  would  have  been 
no  amboceptors  to  unite  the  complement  to  the  bacterial  cells.  The 
complement  would  then  be  at  hand  to  unite  with  the  sensitized  red  cells 
subsequently  added  and  bring  about  their  haemolysis,  as  shown  by  the 
ruby  color  of  the  supernatant  fluid.  This  phenomenon  of  Bordet  and 
Gengou  has  been  utilized  by  Wasserman  for  the  diagnosis  of  diseases 
where  cultures  are  not  applicable.  It  is  in  the  diagnosis  of  syphilis 
that  it  is  best  known.  It  at  present  being  impossible  to  obtain  cultures 
of  Treponema  pallidum,  we  use  emulsions  of  the  liver  of  a  syphilitic 
foetus,  which  have  been  filtered  so  as  to  be  clear,  instead  of  a  culture. 
The  syphilitic  liver,  as  can  be  observed  by  staining  according  to 
Levaditi's  method,  is  packed  with  spirochaetes. 

For  the  immune  bodies  we  take  the  serum  of  the  patient,  or  if  a  case 
of  locomotor  ataxia  or  general  paresis,  the  cerebrospinal  fluid.  This 
is  heated  to  56°  C.  to  destroy  complement  (inactivation).  For  the 
complement  we  use  normal  guinea-pig  serum  in  a  dilution  of  i-io. 
For  the  sensitized  cells  we  use  the  cells  of  some  animal  whose  defibri- 
nated  blood  has  been  used  to  immunize  a  second  animal,  as  human 
blood  injected  into  rabbits.  Consequently,  as  for  haemolysis  experi- 


OPSONIC   POWER.  131 

ments,  we  should  use  a  5%  emulsion  of  human  red  cells  in  salt  solution, 
to  which  has  been  added  the  inactivated  serum  of  the  animal  immune 
to  human  red  cells.  This  immune  serum  should  be  capable  of  dis- 
solving red  cells  in  a  dilution  of  at  least  i  to  1000. 

Experiment. — In  a  test-tube  put  4  drops  of  the  extract  of  syphilitic 
liver  and  the  same  amount  of  the  inactivated  serum  of  the  patient. 
Now  add  i  c.c.  of  a  i-io  dilution  of  guinea-pig  serum  and  allow  the 
mixture  to  remain  in  the  incubator  at  37°  C.  for  i  hour.  Then  add 
i  c.c.  of  the  sensitized  5%  emulsion  of  red  cells,  and  if  haemolysis  does 
not  take  place,  the  patient  has  syphilis.  Controls  should  not  only  be 
made  with  serum  from  normal  persons,  but  also  extracts  prepared  from 
normal  livers  should  be  used  as  well.  This  is  one  of  the  most  exacting 
of  laboratory  diagnostic  tests. 

DETERMINATION  OF  OPSONIC  POWER  AND  THE  PREPARATION 
OF  VACCINES. 

The  following  modification  of  Irishman's  method  takes  very  little 
time  and  skill  and  is  applicable  in  the  determination  of  the  organism 
concerned  in  an  infection,  as  in  Wright's  method.  The  control  of 
vaccine  treatment  by  taking  opsonic  indices  from  time  to  time  does  not 
seem  to  have  met  with  much  favor  in  this  country — the  sources  of  error 
being  as  great,  if  not  greater,  than  ordinary  variations  in  the  opsonic 
index  during  the  negative  and  positive  phases.  Method:  Emulsify, 
by  repeatedly  drawing  up  and  ejecting  with  a  bulb  capillary  pipette,  a 
small  loopful  of  a  young  agar  culture  of  the  organism  to  be  diagnosed 
in  12  to  15  drops  of  salt  solution  containing  i%  of  sodium  citrate  (the 
citrate  prevents  coagulation).  This  may  most  conveniently  be  done 
in  a  watch  glass.  Now  puncture  the  ear  of  the  patient  and  draw  up 
blood  to  a  point  marked  on  the  capillary  bulb  pipette  with  a  grease  pen- 
cil. Draw  in  air  to  make  a  slight  break  in  the  column  and  then  draw 
up  bacterial  emulsion  to  the  same  mark.  Thoroughly  mix  the  blood 
and  emulsion  on  a  slide  by  drawing  up  and  ejecting  the  mixture. 
Then  finally  draw  up  the  mixture  to  about  2  inches  above  the  tip  and 
seal  the  tip  off  in  the  flame.  Put  the  pipette  preparation  in  the  in- 
cubator for  exactly  15  minutes.  Prepare  a  similar  preparation,  using 
the  blood  of  a  normal  person.  At  the  expiration  of  15  minutes'  incu- 


132  PRACTICAL   METHODS    IN   IMMUNITY. 

bation  for  the  patient's  blood  and  the  same  time  for  the  control  blood, 
file  off  the  sealed  tip,  cautiously  eject  the  contents  on  a  slide,  and  after 
mixing,  spread  a  film  on  a  slide  or  cover-glass.  Fix  with  burning 
alcohol  and  stain  with  formoi  fuchsin.  With  Wright's  stain  the 
dark  nucleus  obscures  some  of  the  bacteria.  Counting  the  phago- 
cytized  bacteria  in  a  given  number  of  polymorphonuclears,  we  obtain 
an  average  number  of  bacteria  phagccytized  per  cell.  Repeating  the 
count  with  the  control  or  normal  blood,  we  likewise  have  the  average 
number  of  bacteria  taken  up  per  cell.  Dividing  the  patient's  average 
by  the  normal  average,  we  have  the  opsonic  index.  If  the  average  for 
50  of  the  patient's  cells  was  8  and  that  of  the  control  only  4,  the  patient's 
index  would  be  2,  or  twice  the  normal.  The  practical  value  of  this 
test  is  where  2  or  more  organisms  are  in  a  body  fluid  we  may  ascertain 
the  causative  organism  by  noting  marked  variation  from  the  normal  in 
the  patient's  opsonic  index  for  that  particular  organism  and  not  for  the 
other  organism.  This  variation  may  be  of  the  nature  of  a  high  or  low 
opsonic  index. 

Preparation  of  Vaccines. — It  has  been  found  satisfactory 
to  make  use  of  stock  vaccines  in  gonorrhceal  and  tuberculous  affections. 
In  case  of  other  infections,  however,  and  preferably  with  gonorrhceal 
infections,  the  causative  organism  should  be  isolated  from  pus,  sputum, 
urine,  blood  or  other  material  (autogenous  vaccine).  In  treatment 
of  tuberculosis  Wright  prefers  Koch's  T.  R.  or  Neu  Tuberculin  in 
doses  of  from  1/5000  to  1/800  of  a  milligram.  Some  prefer  Koch's 
more  recent  Bazillen  emulsion.  Having  isolated  the  organism,  it  is 
inoculated  upon  one  or  more  agar  slants,  and  after  a  growth  of  from 
5  to  7  hours  with  streptococci  and  pneumococci,  or  with  18  hours  for 
staphylococci  and  colon,  the  growth  on  these  inoculated  slants  is 
taken  up  with  salt  solution,  thoroughly  shaken  up  in  the  diluting  solu- 
tion and  standardized. 

The  most  practical  way  is  to  gently  rub  off  the  growth  on  the  agar 
in  about  i  or  2  c.c.  of  salt  solution  with  a  platinum  loop.  Then  pour 
the  bacterial  emulsion  into  a  sterile  test-tube  and  repeat  the  process 
with  3  to  5  agar  slants,  until  we  have  from  6  to  10  c.c.  of  the  emulsion 
in  the  sterile  test-tube.  By  heating  to  melting-point  in  the  flame  a  piece 
of  glass  rod  and  attaching  it  to  the  rim  of  the  test-tube  (also  melted), 


PREPARATION   OF    VACCINES.  133 

we  have  a  handle  with  which  to  draw  out  the  test-tube  when  heated 
about  i  inch  from  the  mouth  in  a  blowpipe  flame.  Drawing  this  out, 
we  let  it  cool,  and  then  filing  the  constricted  portion  we  break  it  off 
and  seal  it  in  the  flame.  By  shaking  up  and  down  vigorously  for  5  to 
15  minutes,  the  bacteria  are  distributed  evenly  in  the  salt  solution. 
The  sealed  test-tube  is  then  placed  in  a  water-bath  at  60°  C.  and  heated 
at  this  temperature  for  i  hour.  Again  shake.  The  constricted  sealed 
end  is  again  filed  off  and  a  few  drops  shaken  out  in  a  watch  glass  for 
standardization,  and  at  the  same  time  a  few  drops  are  deposited  on 
an  agar  slant  as  a  test  for  sterility.  (Incubation  for  24  hours  should 
not  show  growth). 

Wright  found  that  by  taking  a  definite  quantity  of  blood  and  a 
similar  quantity  of  bacterial  emulsion,  mixing  the  blood  and  bacterial 
emulsion,  then  making  a  smear  and  staining,  it  was  possible  to  de- 
termine the  ratio  of  bacteria  to  red  cells,  and  from  this  the  number 
of  bacteria  per  cubic  centimeter  could  be  determined.  For  example, 
if  we  find  3  bacteria  to  each  red  cell  we  should  have  15,000,000 
bacteria  to  i  cubic  millimeter.  (There  being  5,000,000  red  cells  to 
the  cubic  millimeter.)  As  one  cubic  centimeter  is  1,000  times  greater 
than  i  cubic  millimeter,  there  would  be  15,000,000,000  bacteria  in 
each  cubic  centimeter  of  such  an  emulsion,  or  vaccine,  as  it  is 
termed.  The  standardization  may  be  made  with  a  haemacytometer.* 

Having  determined  the  strength  of  the  stock  vaccine,  we  should  pre- 
pare a  dilute  vaccine  for  injection.  This  is  most  conveniently  carried 
out  by  filling  vials  with  50  c.c.  of  salt  solution,  plugging  with  cotton, 
then  sterilizing  in  the  autoclave.  A  sterile  rubber  cap  is  now  drawn 
over  the  mouth  of  the  vial.  Sterility  is  insured  by  plunging  the  rubber 
cap  and  neck  in  boiling  water.  If  the  stock  vaccine  showed  5,000,000,- 
ooo  bacteria  per  c.c.  and  we  desired  to  have  a  vaccine  containing  200,- 
000,000  bacteria  per  c.c.,  it  would  only  be  necessary  to  draw  out  2  c.c. 
of  the  salt  solution  by  means  of  a  sterile  syringe  needle  inserted 
through  the  rubber  cap  and  replace  it  with  2  c.c.  of  the  bacterial  emul- 
sion. Example :  In  introducing  2  c.c.  of  a  vaccine  containing  5  billion 

*This  is  best  done  by  drawing  up  the  vaccine  to  .5  with  either  the  red  or  white 
pipette,  according  to  concentration,  and  then  sucking  up  i  to  10  dilute  carbol 
fuchsin  to  1 1  or  101.  Allow  the  bacteria  to  settle  on  the  shelf  for  10  minutes  before 
counting.  Count  as  in  making  a  red  count. 


134  PRACTICAL    METHODS    IN    IMMUNITY. 

bacteria  per  c.c.,  we  throw  in  10  billion  bacteria  in  a  volume  equal  to 
50  c.c.  Then  each  c.c.  of  the  50  c.c.  in  the  bottle  would  contain 
10,000,000,000  divided  by  50  or  200  million  in  each  c.c.  If  we  only 
want  a  vaccine  containing  100  million  per  c.c..  we  should  only  add  i  c.c. 
We  now  add  1/4%  of  Trikresol  to  the  vaccine  in  order  to  insure  ster- 
ility. (Introduced  with  syringe,  inserting  needle  through  rubber  cap.) 
The  syringe  is  best  sterilized  by  drawing  up  vaselin  or  olive  oil  heated 
to  150°  C.,  and  the  neck  and  rubber  cap  of  the  bottle  in  boiling  water. 
We  now  draw  up  the  desired  dose  of  bacteria.  If  glass  syringes  are  used, 
simply  boiling  in  water  suffices.  The  ordinary  doses  are:  For  gono- 
cocci,  streptococci,  pneumococci  and  colon  vaccines  5  million  to  50 
million.  For  staphylococci  200  million  to  one  billion. 


NOTES  ON  BACTERIOLOGY. 


NOTES  ON  BACTERIOLOGY. 


NOTES  ON  BACTERIOLOGY. 


NOTES  ON  BACTERIOLOGY. 


PART  II. 
STUDY  OF  THE  BLOOD. 


CHAPTER  XIII. 
MICROMETRY  AND  BLOOD  PREPARATIONS. 

MlCROMETRY. 

IN  the  examination  of  blood  and  faeces  preparations,  especially 
when  the  identification  of  animal  parasites  is  in  question,  there  is 
nothing  that  assists  more  than  a  knowledge  of  the  measurements  of 
the  object  studied.  The  making  of  such  measurements  microscopic- 
ally is  termed  micrometry. 

Micrometry  is  also  indispensable  in  bacteriology  and  cytodiagnosis 
as  well  as  in  animal  parasitology. 

The  most  practical  way  of  making  these  measurements  is  with  an 
ocular  micrometer.  These  can  be  bought  separately,  or  a  glass  disk 
(disk  micrometer)  with  lines  ruled  on  it  can  be  dropped  into  the  ocular 
to  rest  on  the  diaphragm  inside  the  ocular.  The  ruled  surface  of  this 
glass  diaphragm  should  be  placed  downward.  As  was  stated  in  con- 
nection with  the  microscope,  the  image  of  the  object  is  formed  at  the 
level  of  the  diaphragm  rim  inside  the  ocular,  consequently  the  lines 
of  the  image  cut  those  of  the  lines  ruled  on  the  glass  in  the  ocular. 
Once  having  standardized  the  value  of  the  spaces  of  the  ocular  microm- 
eter for  each  different  objective,  all  that  is  necessary  subsequently  in 
measuring  is  to  count  the  number  of  lines  or  spaces  which  the  image 
of  the  object  fills  and  then,  knowing  the  value  of  each  space  for  that 
objective,  to  multiply  the  number  of  spaces  by  the  value  of  a  single 
space. 

The  unit  in  micrometry  is  the  mikron.     This  is  usually  written  /* 


i36 


MICROMETRY    AND    BLOOD    PREPARATIONS. 


and  is  the  i/iooo  part  of  a  millimeter.      There  are  1000  mikronsina 
millimeter. 

To  standardize:  For  this  purpose  it  is  necessary  to  have  a  scale 
of  known  measurements.  The  stage  micrometers  are  usually  ruled 
in  spaces  of  .1  and  .01  mm.  The  lines  which  are  i/io  of  a  millimeter 


FIG.  48. — Micrometry  diagrams,  i.  Ocular  micrometer  with  stage  micrometer. 
50  spaces  of  ocular  micrometer  cover  two  100  micron  spaces  and  ten  10  micron 
spaces;  equal  300  microns.  Each  division  on  ocular  micrometer  equals  6  microns. 
2.  Ocular  micrometer  subtending  image  of  whip  worm  egg.  9  spaces  of  ocular 
micrometer  cover  Whipworm  egg.  Each  space  equals  6  microns.  Whipworm  egg 
equals  54  microns.  3.  Ocular  micrometer  with  ruling  of  hasmacytometer.  50  spaces 
of  ocular  micrometer  cover  space  equal  to  width  of  6  small  squares  50  x  6  =  300 
microns.  Each  division  of  ocular  micrometer  equals  6  microns. 


apart  are  consequently  separated  by  a  distance  of  loomikrons;  those 
i/ 100  of  a  millimeter  apart  are  separated  by  a  distance  of  10  mikrons. 

The  ocular  micrometer  is  usually  ruled  with  50  or  100  lines  or 
spaces,  separated  by  longer  lines  into  groups  of  5  and  10. 

Having  brought  the  lines  on  the  stage  micrometer  to  a  focus,  we 


MICROMETRY.  137 

determine  the  number  of  spaces  on  the  stage  micrometer  which  the 
100  divisions  of  the  ocular  micrometer  cover.  To  distinguish  the 
ruling  of  the  ocular  from  that  of  the  stage  micrometer,  revolve  the 
ocular  with  the  fingers. 

The  tube  length  which  is  used  at  the  time  of  standardizing  must 
always  be  adhered  to  in  subsequent  measurements. 

Example:  With  a  2/3-in.  objective,  the  100  rulings  of  the  ocular 
fill  in  15  of  the  i/io  millimeter  rulings  (ioo//)  and  3  of  the  i/ioo 
millimeter  spaces  (lOfi).  Consequently  the  100  spaces  of  the  ocular 
cover  1530  mikrons  (15  x  100  =  1500;  3  x  10  =  30).  Then  if  100 
spaces  equal  1530  mikrons,  one  space  would  equal  15.3  mikrons.  With 
the  i/6-in.  objective  the  100  ocular  spaces  would  cover  about  3  of  the 
i/ 10  millimeter  (ioo(«)  spaces  of  the  stage  micrometer.  Then  the 
100  spaces  would  equal  300  mikrons  and  one  space  would  equal  3 
mikrons. 

The  ruling  of  the  slide  of  a  Thoma  Zeiss  haemacytometer  will 
answer  as  well  as  a  stage  micrometer.  The  small  squares  are  1/20  of  a 
millimeter  square,  consequently  the  distance  between  the  lines  border- 
ing the  small  square  is  1/20  millimeter  or  50  mikrons. 

Now,  if  wifh  the  1/6  in.  objective,  the  100  lines  on  the  ocular  fill 
in  the  spaces  of  6  small  squares,  the  length  of  such  a  space  would  be 
50  x  6  =  300  mikrons.  This  divided  by  100  spaces  would  equal  3  ,«. 

The  most  accurate  instrument  for  measuring  is  the  filar  microm- 
eter. These  are  expensive.  Measurements  can  also  be  made  with 
the  camera  lucida,  but  it  takes  considerable  time  to  make  the  adjust- 
ments necessary,  so  that  it  is  not  convenient.  With  an  ocular  microm- 
eter one  can  make  measurements  of  blood-cells,  amoebae,  etc.,  in  a 
few  seconds — it  only  being  necessary  to  slip  in  the  ocular  micrometer. 

Rule  for  determining  the  magnifying  power  of  microscopic  lenses: 
Measure  the  diameter  of  the  lens  of  the  objective  in  inches — the 
approximate  equivalent  focal  distance  is  about  twice  the  diameter. 
Dividing  10  by  the  equivalent  focal  distance  gives  the  magnifying 
power  of  the  lens.  This  should  be  multiplied  by  the  number  of  times 
the  ocular  magnifies.  Example:  The  diameter  of  the  lens  of  the 
objective  was  found  to  measure  1/2  in.,  the  focal  distance  would 
then  be  about  i  inch.  Dividing  10  by  i  we  have  10  as  the  magnifying 


138  MICROMETRY    AND    BLOOD    PREPARATIONS. 

power  of  the  lens  of  the  objective.     If  we  were  using  a  No.  4  ocular, 
the  magnifying  power  would  be  approximately  40. 

BLOOD  PREPARATIONS. 

To  obtain  blood,  except  for  blood  cultures,  use  either  a  platino- 
iridium  hypodermic  needle  which  can  be  sterilized  in  the  flame,  a 
small  lancet  or  a  surgical  needle  with  cutting  edge.  A  steel  pen  with 
one  nib  broken  off  or  the  glass  needle  of  Wright  may  also  be  used. 
To  make  a  glass  needle,  pull  straight  apart  a  piece  of  capillary  tubing 
in  a  very  small  flame.  Tap  the  fine  point  to  break  off  the  very  delicate 
extremity.  Scarcely  any  pain  attends  the  use  of  such  a  needle.  In 
puncturing  either  the  tip  of  the  finger  or  lobule  of  the  ear  a  quick  piano- 
touch-like  stroke  should  be  used.  The  ear  is  preferable,  as  it  is  less 
sensitive  and  there  is  less  danger  of  infection.  Before  puncturing,  the 
skin  should  be  cleaned  with  70%  alcohol  and  allowed  to  dry.  It  is 
advisable  to  sterilize  the  needle  before  using  it. 

The  first  drop  of  blocd  which  exudes  should  be  taken  up  on  the 
paper  of  the  Ta'lquist  haemoglobinometer,  using  subsequent  ones  for 
the  blood  pipettes  and  smears.  If  it  is  necessary  to  make  a  complete 
blood  examination,  it  is  rather  difficult  to  draw  up  the  blood  in  the 
pipettes,  dilute  it  and  then  get  material  for  fresh  blood  preparations 
and  films  without  undue  squeezing,  which  is  to  be  avoided.  Of 
course,  fresh  punctures  can  be  made.  Ordinarily,  complete  blood 
examinations  are  not  called  for.  It  is  only  a  white  count  or  a  differ- 
ential count  or  an  examination  for  malaria  that  is  required. 

HEMOGLOBIN  ESTIMATION. 

The  most  accurate  instrument  for  this  purpose  is  the  Miescher 
modification  of  the  v.  Fleischl  haemoglobinometer.  The  glass  wedge 
for  comparison  with  the  diluted  blood  is  the  same  in  each  instrument, 
but  by  the  use  of  a  diluting  pipette  accurate  dilutions  are  possible 
in  the  Miescher.  There  are  two  cells  provided — one  12  millimeters 
high,  the  other  15  millimeters;  the  idea  of  this  being  to  enable  one  to 
make  separate  comparisons  and  to  select  the  central  parts  of  the 
glass-wedge  scale,  where  comparison  is  more  accurate  than  at  the  ends. 

If  using  a  i  to  200  dilution  and  the  deeper  cell,  the  reading  of  the 


HEMOGLOBIN    ESTIMATION. 


139 


scale  is  the  proper  one;  if  the  12  millimeter  cell  is  used,  the  reading 
is  only  4/5  of  what  it  should  be.  Thus  a  reading  of  100%  with  the  15- 
millimeter  cell  would  show  with  the  12-millimeter  one  a  reading  of 
80%  for  the  same  blood.  The  apparatus  is  quite  expensive  and 
requires  considerable  time  in  making  the  estimation. 

Sahli's  Haemometer.— A  simple  and  ap- 
parently very  scientific  instrument  which  has 
been  recently  introduced  is  the  Sahli  modifi- 
cation of  the  Gower  haemoglobinometer.  In- 
stead of  the  tinted  glass,  or  gelatin  colored 
with  picrocarmine  to  resemble  a  definite  blood 
dilution,  Sahli  uses  as  a  standard  the  same 
corloring  matter  as  is  present  in  the  tube 
containing  the  blood.  By  acting  on  blood 
with  10  times  its  volume  of  N/io  HC1, 
haematin  hydrochlorate  is  produced,  which 
gives  a  brownish-yellow  color.  In  the 
standard  tube,  which  is  sealed,  a  dilution 
representing  i%  of  normal  blood  is  used. 
To  apply  this  test,  pour  in  N/ 10  HC1  to  the 
mark  10  on  the  scale  of  the  graduated  tube. 
Add  to  this  20  cubic  millimeters  of  the  blood 
to  be  examined,  drawn  up  by  the  capillary 
pipette  provided.  So  soon  as  the  mixture 
assumes  a  clear  brown  color,  add  water  drop 
by  drop  until  the  color  of  the  tubes  matches. 
The  reading  of  the  height  of  the  aqueous 
dilution  on  the  scale  gives  the  Hb.  reading. 
The  tubes  are  encased  in  a  vulcanite  frame 
with  rectangular  apertures.  This  gives  the 
same  optical  impression  as  would  piano -parallel  glass  sides.  It 
recommended  that  the  N/io  HC1  be  preserved  with  chloroform. 

Tallquist's  Haemoglobin  Scale.— This  is  a  small  book  of  specially 
prepared  filter-paper  with  a  color-scale  plate  of  10  shades  of  blood 
colors.  These  are  so  tinted  as  to  match  blood  taken  up  on  a  piece 
of  the  filter-paper  and  are  graded  from  10  to  100.  So  soon  as  the 


FIG.  49. — Sahli's   haemo- 
globinometer.     (Greene.) 


is 


140 


MICROMETRY   AND    BLOOD    PREPARATIONS. 


blood  on  the  filter-paper  has  lost  its  humid  gloss,  the  comparison 
should  be  made.  This  is  best  done  by  shifting  the  blood  stained  piece 
of  filter-paper  suddenly  from  one  to  the  other  of  the  holes  cut  in  each 
shade — the  piece  of  filter-paper  being  underneath  the  color  plate. 
The  error  with  this  method  is  probably  not  over  10%  after  a  little 
experience.  If  the  colored  plate  is  not  kept  in  the  dark,  the  tints  tend 
to  fade.  The  use  of  this  method  requires  neither  time  nor  trouble. 

To  COUNT  BLOOD-CORPUSCLES. 

The    instrument    almost    universally  used    is    the    Thoma-Zeiss 

haemacytometer.  The  apparatus  consists  of  two  pipettes,  one  for 

leukocytes,  graduated  to  give  a  dilution 
of  i  to  10  or  greater;  the  other,  for  red 
cells  to  give  a  dilution  of  i  to  100  or 
greater.  The  white  pipette  has  the  mark 
ii  above  the  bulb  and  the  red  pipette 
the  mark  101.  In  addition,  there  is  a 
counting  chamber.  This  consists  of  a 
square  of  glass  with  a  round  hole  in  the 
center.  Occupying  the  center  of  this 
round  hole  is  a  circular  disk  of  glass  of 
less  diameter,  so  that  an  encircling 
channel  is  left.  The  square  and  the 
FIG.  50. — Thoma-Zeiss  blood  circle  of  glass  are  cemented  to  a  heavy 

counter,  showing  pipette   count-       lags    siide        The   surfaces   of    each    are 

mg    chamber    and    ruled   field. 

(Greene.}  absolutely   level    and    highly    polished. 

That  of  the  circular  disk  is  ruled  into 

squares  of  varying  size  and  is  exactly  i/io  of  a  millimeter  below  the 
level  of  the  surface  of  the  surrounding  glass  square. 

When  a  polished  piano-parallel  cover-glass  rests  on  the  shelf,  as 
the  outer  square  glass  is  termed,  there  is  a  space  left  between  its  under 
surface  and  the  ruled  disk  of  .1  millimeter.  The  channel  around  the 
disk  is  termed  the  moat  or  ditch.  The  most  desirable  rulings  are 
those  of  Turck  and  of  Zappert.  In  these  the  entire  ruled  surface 
consists  of  9  large  squares,  each  i  millimeter  square.  These  are 
subdivided,  and  in  the  central  large  square  are  to  be  found  the  small 


COUNTING   RED   CELLS.  141 

squares  used  for  averaging  the  red  cells.  These  small  squares  are 
1/20  of  a  millimeter  square  and  are  arranged  in  9  groups  of  16  small 
squares  by  bordering  double-ruled  lines.  As  the  unit  in  blood  counting 
is  the  cubic  millimeter,  if  one  counted  all  the  white  cells  lying  within 
one  of  the  large  squares  (i  millimeter  square),  he  would  have  only 
counted  the  cells  in  a  layer  i/io  of  the  required  depth,  so  that  it  would 
be  necessary  to  multiply  the  number  obtained  by  10.  This  product, 
multiplied  by  the  dilution  of  the  blood,  would  give  the  number  of 
white  cells  in  a  cubic  millimeter  of  undiluted  blood. 

To  make  a  red  count :  Having  a  fairly  large  drop  of  blood,  apply  the 
tip  of  the  10 1  pipette  to  it  and,  holding  the  pipette  horizontally,  care- 
fully and  slowly  draw  up  with  suction  on  the  rubber  tube  a  column  of 
blood  to  exactly  .5  or  i.  The  variation  of  1/25  of  an  inch  from  the 
mark  would  make  a  difference  of  almost  3  percent.  If  the  column 
goes  above  .5,  it  can  be  gently  tapped  down  on  a  piece  of  filter-paper 
until  the  .5  line  is  cut.  Now  insert  the  tip  of  the  pipette  into  some 
diluting  fluid  and,  revolving  the  pipette  on  its  long  axis  while  filling  it 
by  suction,  you  continue  until  the  mark  101  is  reached.  A  variation  of 
1/25  of  an  inch  at  this  mark  would  only  give  an  error  of  about  1/30  of 
i%.  After  mixing  thoroughly,  by  shaking  for  one  or  two  minutes,  the 
fluid  in  the  pipette  below  the  bulb  is  expelled  (this,  of  coarse,  is  only 
diluting  fluid).  A  drop  of  the  diluted  blood  of  a  size  just  sufficient  to 
cover  the  disk  when  the  cover-glass  is  adjusted,  is  then  deposited  on  the 
disk  and  the  cover-glass  applied  by  a  sort  of  sliding  movement,  best 
obtained  by  using  a  forceps  in  one  hand  assisted  by  the  thumb  and 
index-finger  of  the  other. 

Among  diluting  fluids  Toisson's  is  probably  the  best  : 

Sodium  chloride,  i  gram. 

Sodium  sulphate,  8  grams. 

Glycerin,  30  c.c. 

Distilled  water,  160  c.c. 

Dissolve  the  sodium  chloride  and  the  sodium  sulphate  in  the 
glycerin  water  and  add  sufficient  methyl  or  gentian  violet  to  give  a 
rich  violet  tint. 

A  salt  solution  of  about  2%  strength,  tinged  with  about  i  drop  of  a 


142  MICROMETRY    AND    BLOOD    PREPARATIONS. 

saturated  alcoholic  solution  of  gentian  violet  to  about  50  c.c.,  is  a  good 
substitute,  or  the  salt  solution  alone  will  answer  when  no  white  count  is 
to  be  made  at  the  same  time  as  the  red  one. 

It  is  important  to  work  quickly  in  adjusting  the  cover-glass,  or 
there  will  be  cells  settling  in  the  center  of  the  drop  from  a  greater  depth 
than  the  one  which  the  apposition  of  the  cover-glass  makes  (i/io 
millimeter  deep). 

A  good  preparation  should  show: 

1.  Presence  of  Newton's  rings. 

2.  Absence  of  air  bubbles. 

3.  Entire  surface  of  ruled  disc  covered. 

4.  Equal  distribution  of  cells. 

Before  counting,  about  5  minutes  should  be  allowed  for  the  settling 
of  the  cells. 

It  will  be  remembered  that  the  small  squares  are  1/20  millimeter 
square.  The  depth  of  fluid  from  upper  surface  of  shelf  to  lower  sur- 
face of  cover-glass  is  i/io  mm.  Hence  each  space  embraced  by  the 
small  square  and  the  depth  of  fluid  is  1/4000  of  the  unit  used  in  esti- 
mating number  of  corpuscles  in  blood,  or  i  cubic  millimeter  (1/20x1/20 
x  1/10  =  1/4000).  Count  100  of  the  small  squares  (this  enables  one 
to  use  decimals).  There  are  9  squares  between  double -ruled  lines, 
each  containing  16  small  squares.  Count  the  number  of  corpuscles  in 
the  1 6  small  squares  contained  in  upper  left-hand  double-ruled  square. 
Put  down  this  count.  Next  count  corpuscles  in  the  adjoining  16 
squares.  Put  down  this  count.  Then  in  third,  16  squares.  Put 
down  the  number.  Now  move  down  to  next  row  of  three  double- 
ruled  squares.  Count  the  number  of  corpuscles  in  each  of  the  three 
1 6  square  spaces  and  set  down  the  numbers  for  addition.  We  have  now 
counted  96  small  squares  (6  x  16).  Count  at  any  place  4  additional 
small  squares  and  add  number  of  blood -cells  contained  therein  to  those 
in  the  96  small  squares  already  counted.  Divide  the  sum  by  100  or 
simply  point  off  two  decimals.  This  gives  the  average  for  each  small 
square.  Multiply  this  by  the  dilution  and  then  (as  the  small  square 
is  only  1/4000  cu.  mm.)  by  4000.  This  will  give  the  number  of  cor- 
puscles in  i  cubic  millimeter.  Example:  100  small  squares  contained 
655  red  cells.  Pointing  off  6.55  equals  average  number  of  red  cells  per 


LENCOCYTE    COUNTING.  143 

small  square.  Multiply  by  dilution  (200)  and  then  by  4000  (the  small 
square  is  4000  times  smaller  than  the  unit:  icu.  mm.) — 6.55x200  = 
1310  x  4000  =  5,240,000. 

At  least  100  small  squares,  and  preferably  200  should  be  counted. 
If  the  blood  appears  normal,  one  may  simply  count  the  number  of  red 
cells  in  5  of  the  16  small  square  spaces  (80  small  squares).  Having 
added  the  numbers  and  multiplying  by  10,000,  you  obtain  the  number 
of  cells  in  i  cubic  millimeter.  (Eighty  small  squares  is  1/50  of  the 
unit  of  i  cu.  mm.,  or  4000  small  squares.  The  blood  dilution  being 
i  to  200,  we  have  50  x  200  x  number  of  cells  in  80  small  squares.) 

In  counting,  count  corpuscles  lying  on  the  lines  above  and  to  the 
right.  Do  not  count  those  lying  on  lines  below  and  to  the  left. 

In  the  small  squares  count  only  corpuscles  lying  in  the  space  or 
cutting  the  upper  line.  This  prevents  counting  the  same  cell  twice. 

To  Count  White  Cells.— Draw  up  the  fluid  in  the  white  pipette  to 
the  mark  .5.  Then,  still  holding  the  pipette  as  near  the  horizontal 
as  possible,  because  the  column  of  blood  tends  to  fall  down  in  the 
larger  bore,  draw  up  by  suction  a  diluting  fluid  which  will  disintegrate 
the  red  cells  without  injuring  the  whites.  The  best  fluid  is  .3%  of 
glacial  acetic  acid  in  water.  This  makes  the  white  cells  stand  out  as 
highly  refractile  bodies.  Some  prefer  to  tinge  the  fluid  with  gentian 
violet.  The  .5  mark  is  preferred  because  it  takes  a  very  large  drop  of 
blood  to  fill  the  tube  up  to  the  i  mark  and  if  there  is  much  of  a  leukocy- 
tosis  a  i  to  10  dilution  is  not  sufficient.  In  leukemic  blood  it  is  better 
to  use  the  red  pipette  with  the  .3%  acetic  acid  solution 

The  blood  having  been  drawn  up  to  .5,  we  have  a  dilution  of  i  to  20. 
Making  a  preparation,  exactly  as  was  done  in  the  case  of  the  red  count, 
we  count  all  of  the  white  cells  in  one  of  the  large  squares  (i  sq.  mm.). 
The  cross  ruling  greatly  facilitates  this.  Note  the  number.  Then 
count  a  second  and  a  third  large  square.  Strike  an  average  for  the 
large  squares  counted  and  multiply  this  by  10,  as  the  depth  of  the 
fluid  gives  a  content  equal  to  only  i/io  of  a  cubic  millimeter.  Then 
multiply  by  the  dilution.  Example:  First  large  square  50;  second 
large  square  70;  third  large  square  60.  Average  60.  Then  60  x  10  x 
20  =  12,000,  the  number  of  leukocytes  in  i  cubic  millimeter  of  blood. 
The  count  may  be  made  with  a  low  power  (2/3-in.  objective)  as  the 


144  MICROMETRY   AND    BLOOD    PREPARATIONS. 

leukocytes  stand  out  like  pearls.  It  is  better,  however,  to  use  a  higher 
power,  so  that  pieces  of  foreign  material  may  be  recognized  and  not 
enumerated  as  white  cells. 

When  it  is  desired  to  make  a  white  count  with  the  same  preparation 
as  is  used  for  the  red  one,  especially  if  the  ruling  is  of  the  old  style 
(only  central  ruling  and  not  in  9  large  squares  as  with  Zappert  and 
Turck),  it  is  advisable  to  make  use  of  the  method  of  counting  by  fields. 
With  a  Leitz  No.  4  ocular  and  a  No.  6  objective,  with  a  tube  length  of 
120  millimeters,  it  will  be  observed  that  the  field  so  obtained  has  a 
diameter  of  8  small  squares.  Now,  remembering  that  the  area  of  a 
circle  equals  the  square  of  the  radius  multiplied  by  *-,  or  3.1416,  we 
have  the  following  calculation:  The  diameter  being  8  small  squares, 
the  radius  would  be  4  small  squares.  Squaring  the  radius,  we  have 
1 6.  This  multiplied  by  3.1416  gives  us  50.  This  means  that  every 
field,  with  the  microscope  adjusted  as  stated,  contains  50  of  the 
small  squares,  or  1/80  of  the  unit  of  i  cubic  millimeter  of  the  diluted 
blood. 

By  keeping  a  single  red  cell  in  view  while  moving  the  mechanical 
stage  from  right  to  left  or  from  above  downward,  we  know  that  a  new 
field  of  50  small  squares  is  brought  into  view  when  the  circumference 
of  the  field  cuts  this  individual  cell.  Example:  As  2000  small  squares 
would  ordinarily  be  a  sufficient  number  to  count  for  a  white  count, 
this  would  require  us  to  count  the  number  of  leukocytes  in  40  of  the 
designated  microscopic  fields  (this,  of  course,  is  only  1/2  the  unit, 
hence  we  should  multiply  by  2).  Counted  40  fields  and  noted  50 
white  cells.  50  x  2  =  100  x  200  (the  dilution  in  red  pipette)  =  20,000. 
Consequently  20,000  would  represent  the  number  of  leukocytes  in  i 
cubic  millimeter  of  the  blood  examined. 

After  making  a  blood  count,  the  haemacytometer  slide  should  be 
cleaned  with  soap  and  water  and  then  rubbed  dry,  preferably  with  an 
old  piece  of  linen.  As  the  accuracy  of  the  counting  chamber  depends 
upon  the  integrity  of  the  cement,  any  reagent  such  as  alcohol,  xylol, 
etc.,  and  in  particular  heat,  will  ruin  the  instrument.  The  pipettes 
should  be  cleaned  by  inserting  the  ends  into  the  tube  from  a  vacuum 
pump,  as  a  Chapman  pump.  First  draw  water  or  i%  sod.  carbonate 
solution  through  the  pipette,  then  alcohol,  then  ether,  and  finally 


FRESH    BLOOD    FILMS.  145 

allow  air  to  pass  through  to  dry  the  interior.  If  the  interior  is  stained, 
use  i%  HC1  in  alcohol.  If  a  vacuum  pump  is  not  at  hand,  a  bicycle 
pump  or  suction  by  mouth  will  answer. 

PREPARATIONS  FOR  THE  STUDY  OF  FRESH  BLOOD. 

Many  authorities  prefer  a  fresh-blood  specimen  to  a  stained  dried 
smear  in  the  study  of  parasites  of  the  blood.  In  malaria  in  particular 
there  is  so  much  information  as  to  species  to  be  obtained  from  a  fresh 
specimen  that  the  employment  of  this  method  should  never  be  ne- 
glected. While  waiting  for  the  film  to  stain  one  has  5  or  6  minutes 
which  could  not  be  better  spent  than  in  examining  the  fresh  specimen 
which  only  requires  a  moment  to  make. 

Manson's  Method. — Have  a  perfectly  clean  cover-glass  and  slide. 
Touch  the  apex  of  the  exuding  drop  of  blood  with  the  cover-glass  and 
drop  it  on  the  center  of  the  slide.  The  blood  flows  out  in  a  film  which 
exhibits  an  "empty  zone"  in  the  center.  Surrounding  this  we  have 
the  "zone  of  scattered  corpuscles, " next  the  "single  layer  zone"  and 
the  "zone  of  rouleaux"  at  the  periphery.  It  is  well  to  ring  the  prepara- 
tion with  vaselin.  When  desiring  to  demonstrate  the  flagellated 
bodies  in  malaria,  it  is  well  to  breathe  on  the  cover-glass  just  prior  to 
touching  the  drop  of  blood. 

The  Method  of  Ross  is  very  easy  of  application  and  gives  most 
satisfactory  preparations.  Take  a  perfectly  clean  slide  and  make  a 
vaselin  ring  or  square  of  the  size  of  the  cover-glass.  Then,  having 
taken  up  the  blood  on  the  cover-glass,  drop  it  so  that  its  margin  rests  on 
the  vaselin  ring.  Gently  pressing  down  the  cover-glass  on  the  vase- 
lin makes  beautiful  preparations  wrhich  keep  for  a  very  long  time.  If 
it  is  desired  to  study  the  action  of  stains  on  living  cells,  this  method  is 
also  applicable.  A  very  practical  way  to  do  this  is  to  tinge  .85%  salt 
solution  containing  i%  sodium  citrate  (the  same  as  is  used  in  opsonic 
work)  with  methylene  azur,  gentian  violet  or  methyl  green.  With  a 
Wright  bulb  pipette,  take  up  one  part  of  blood,  then  one  part  of  tinted 
salt  solution.  Mix  them  quickly  on  a  slide  and  then  deposit  a  small 
drop  of  the  mixture  in  the  center  of  the  vaselin  ring  and  immediately 
apply  a  cover-glass  and  press  down  the  margins  as  before.  This  method 
will  be  found  of  great  practical  value. 


146 


MICROMETRY    AND    BLOOD    PREPARATIONS 


PREPARATION  AND  STAINING  OF  DRIED  FILMS. 
When  preparations  are  desired  for  a  differential  count,  Ehrlich's 
method  of  making  films  is  to  be  preferred  as  the  different  types  of 
leukocytes  are  more  evenly  distributed.  In  making  smears  by  spread- 
ing, there  is  a  tendency  for  the  polymorphonuclears  to  be  concentrated 
at  the  margin  while  lymphocytes  remain  in  the  central  part  of  the  film. 


FIG.  51. — Blood  technic.  i,  2,  3,  Method  for  making  blood  smear  on  slide; 
4,  U  tube  for  resting  slides  while  staining;  5,  slide  showing  grease  pencil  marking, 
marking  prevents  stain  from  overflowing;  6,  method  for  drawing  apart  cover 
glasses  in  making  blood  smear. 

In  Ehrlich's  method  we  have  perfectly  clean  dry  cover-slips.  Take 
up  a  small  drop  of  blood  without  touching  the  surface  of  the  ear  or  finger. 
Drop  this  cover-glass  immediately  on  a  second  one  and  as  soon  as  the 
blood  runs  out  in  a  film,  draw  the  two  cover-slips  apart  in  a  plane 
parallel  to  the  cover-glasses.  Slide  them  apart.  Ehrlich  uses  forceps 
to  hold  the  cover-glasses  to  avoid  moisture  from  the  fingers. 

Of  the  various  methods  of  spreading  films  on  slides  there  is  none 


BLOOD   FILMS.  147 

equal  to  that  described  by  Daniels.  In  this  the  drop  of  blood  is  drawn 
along  and  not  pushed. along.  The  films  are  even,  can  be  made  of  any 
desired  thickness  by  changing  the  angle  of  the  drawing  slide,  and  there 
is  little  liability  of  crushing  pathological  cells.  Take  a  small  drop  of 
blood  on  the  end  of  a  clean  slide.  Touch  a  second  slide  about  1/2 
inch  from  end  with  the  drop  and  as  soon  as  the  blood  runs  out  along 
the  line  of  the  slide  end,  slide  it  at  an  angle  of  45°  to  the  other  end  of 
the  horizontal  slide.  The  blood  is  pulled  or  drawn  behind  the  ad- 
vancing edge  of  the  advancing  slide.  An  angle  less  than  45°  makes  a 
thinner  film.  One  greater,  a  thicker  film. 

Of  the  various  methods  of  making  smears  by  means  of  cigarette 
paper,  rubber  tissue,  needles,  etc.,  the  best  seems  to  be  to  take  a  piece  of 
capillary  glass  tubing  and  use  this  instead  of  a  needle  in  making  the 
film.  There  is  one  advantage  about  the  strip  of  cigarette  paper 
touched  to  the  drop  of  blood  and  drawn  out  along  the  slide  or  cover 
glass  and  that  is  that  it  is  almost  impossible  not  to  make  a  working 
preparation  by  this  method. 

In  the  making  of  smears  the  chief  points  are  to  make  the  smear  as 
soon  after  taking  the  blood  as  possible  and  to  have  slides  and  cover- 
glasses  scrupulously  clean.  It  is  well  to  flame  all  slides  and  cover- 
glasses  which  are  to  be  used  for  blood-work.  This  is  the  best  method 
of  getting  rid  of  grease. 

Fixation  of  Film. — In  Wright's,  Irishman's  and  other  similar 
stains  the  methyl-alcohol  solvent  causes  the  fixation.  In  staining 
with  Giemsa's  stain,  Ehrlich's  tri-acid,  haematoxylin  and  eosin, 
Smith's  formol  fuchsin,  and  with  thionin,  separate  fixation  is  necessary. 
For  Giemsa  and  thionin,  either  alcohol  and  ether  (15  minutes),  ab- 
solute alcohol  (10  to  15  minutes)  or  methyl  alcohol  (2  to  5  minutes) 
answer  well. 

Formalin  vapor,  for  5  to  10  seconds,  is  also  used  for  fixation.  For 
Ehrlich's  tri-acid,  haematoxylin  and  eosin  and  formol  fuchsin,  heat 
gives  the  best  results.  The  best  method  is  to  place  the  films  in  an 
oven  provided  with  a  thermometer.  Raise  the  temperature  of  the 
oven  to  135°  C.  and  then  remove  the  burner.  After  the  oven  has 
cooled,  take  out  the  fixed  slides  or  slips. 

Some  prefer  to  place  a  crystal  of  urea  on  the  slide,  then  hold  it  over 


148  MICROMETRY   AND    BLOOD    PREPARATIONS. 

the  flame  until  the  urea  melts.     This  shows  that  a  temperature  be- 
tween 130°  and  135°  C.  has  been  reached. 

One  of  the  handiest  methods  is  to  drop  a  few  drops  of  95%  alcohol 
on  the  slide  or  cover  glass.  Allow  this  to  flow  over  the  entire  surface; 
then  get  rid  of  the  excess  of  alcohol  by  touching  the  edge  to  a  piece  o£. 
filter  paper  for  a  second  or  two.  Then  light  the  remaining  alcohol 
film  from  the  flame  and  allow  the  burning  alcohol  to  burn  itself  out. 
A  chemical  fixation  which  gives  good  fixation  for  haematoxylin  and 
tri-acid  stains  (not  equal  to  heat)  is  a  modification  of  Zenker's  fluid 
(Whitney).  To  Muller's  fluid,  which  is  potassium  bichromate  2  gms., 
sodium  sulphate  i  gm.  and  water  100  c.c.,  add  5  gms.  of  bichloride  of 
mercury  and  5  c.c.  of  nitric  acid  (C.  P.).  Fixation  is  obtained  in  5 
seconds. 

Staining  Blood-films. — As  separate  staining  with  eosin  and 
methylene  blue  rarely  gives  good  preparations  and  as  the  modifications 
of  the  Romanowsky  stain  recommended  are  easy  to  make  and  employ, 
and  give  much  greater  information,  the  separate  method  of  staining  is 
not  recommended.  The  most  satisfactory  single  stain  is  thionin. 

Rees'  Thionin  Solution. — Take  of  thionin  1.5  gms.,  alcohol  10  c.c., 
aqueous  solution  of  carbolic  acid  (5%)  100  c.c.  Keep  this  as  a  stock 
solution.  It  should  be  at  least  two  weeks  old  before  using.  For  use, 
filter  off  5  c.c.  and  make  up  to  20  c.c.  with  water. 

1.  Fix  films  (a)  by  heat,  (b)  by  alcohol  and  ether,  or  (c)  preferably 
by  i%  formalin  in  95%  alcohol  for  i  minute. 

2.  Stain  for  from  10  to  20  minutes.     Wash  and  mount.     Malarial 
parasites  are  stained  purplish;  nuclei  of  leukocytes,  blue;  red  cells, 
faint  greenish-blue. 

Ehrlich's  Tri-acid  or  Triple  Stain. — There  are  required : 

1.  Sat.  aq.  sol.  orange  G.     (Dissolve  3  grams  in  50  c.c.  water.) 

2.  Sat.  aq.  sol.  acid  fuchsin.    (Dissolve  10  grams  in  50  c.c.  water.) 

3.  Sat.  aq.  sol.  methyl  green.     (Dissolve    10    grams    in   50  c.c. 
water.) 

These  three  solutions  may  be  kept  as  stock  solutions.  They  keep 
well  in  the  dark.  To  make  the  stain:  Add  9  c.c.  of  No.  2  (acid 
fuchsin)  to  18  c.c.  of  No.  i  (orange  G.).  After  they  are  mixed 
thoroughly,  add  20  c.c.  of  No.  3  (methyl  green).  Then,  after  the  first 


ROMANOWSKY   METHODS   OF    STAINING.  149 

three  ingredients  are  well  mixed,  add  5  c.c.  of  glycerin.  Mix,  then  add 
15  c.c.  of  alcohol;  again  mix,  and  finally  add  30  c.c.  of  distilled  water. 
Keep  the  mixed  stain  about  i  week  before  using.  The  best  fixatives 
are  heat  and  Whitney's  modified  Zenker.  To  use:  Stain  films  from 
2  to  5  minutes.  Then  wash  and  mount.  The  tri-acid  stain  is  a  good 
tissue  stain.  The  objections  to  the  triacid  stain  are  that  it  does  not 
stain  malarial  parasites  or  mast  cells  and  that  failure  to  obtain  good 
results  is  of  frequent  occurrence. 

Wright's  Method. — The  stain  is  made  by  adding  r  gram  of  methylene 
blue  (Grubler)  to  100  c.c.  of  a  1/2%  solution  of  sodium  bicarbonate  in 
water.  This  mixture  is  heated  for  i  hour  in  an  Arnold  sterilizer. 
When  cool,  add  to  the  methylene-blue  solution  500  c.c.  of  a  i  to  1000 
eosin  solution  (yellow  eosin,  water  soluble).  Add  the  eosin  solution 
slowly,  stirring  constantly  until  the  blue  color  is  lost  and  the  mixture 
becomes  purple  with  a  yellow  metallic  luster  on  the  surface  and  there 
is  formed  a  finely  granular  black  precipitate.  Collect  this  precipitate 
on  a  filter-paper  and  when  thoroughly  dry  (dry  in  the  incubator  at 
38°  C.)  dissolve  .3  gm.  in  100  c.c.  of  pure  methyl  alcohol  (acetone free). 
This  constitutes  the  stock  solution.  For  use  filter  off  20  c.c.  and  add  to 
the  filtrate  5  c.c.  of  methyl  alcohol. 

A  modification  by  Batch  is  very  satisfactory.  In  this  method  in- 
stead of  polychroming  the  methylene  blue  with  sodium  bicarbonate 
and  heat,  the  method  of  Borrel  is  used.  Dissolve  i  gm.  of  methylene 
blue  in  100  c.c.  of  distilled  water.  Next  dissolve  .5  gm.  of  silver 
nitrate  in  50  c.c.  of  distilled  water.  To  the  silver  solution  add  a  2  to 
5%  caustic  soda  solution  until  the  silver  oxide  is  completely  precipi- 
tated. Wrash  the  precipitated  silver  oxide  several  times  with  distilled 
water.  This  is  best  accomplished  by  pouring  the  wash-water  on  the 
heavy  black  precipitate  in  the  flask,  agitating,  then  decanting  and 
again  pouring  on  water.  After  removing  all  excess  of  alkali  by 
repeated  washings,  add  the  methylene-blue  solution  to.  the  precipitated 
silver  oxide  in  the  flask.  Allow  to  stand  about  10  days,  occasionally 
shaking  until  a  purplish  color  develops.  The  process  may  be  hastened 
in  an  incubator.  When  polychroming  is  complete,  filter  off  and  add 
to  the  filtrate  the  i  to  1000  eosin  solution  and  proceed  exactly  as  with 
Wright's  stain. 


150  MICROMETRY    AND    BLOOD    PREPARATIONS 

In  Leishman's  method  the  polychroming  is  accomplished  by  adding 
i  gm.  of  methylene  blue  to  100  c.c.  of  a  1/2%  solution  of  sodium  car- 
bonate. This  is  kept  at  65°  C.  for  12  hours  and  allowed  to  stand  at 
room  temperature  for  10  days  before  the  eosin  solution  is  added.  The 
succeeding  steps  are  as  for  Wright's  stain. 

The  modification  of  the  Romanowsky  stain,  which  is  used  in  the 
laboratory  of  the  U.  S.  Naval  Medical  School  and  which  can  be  rec- 
ommended as  giving  good  results  with  the  least  expenditure  of  time 
in  making  and  which  by  the  addition  of  either  acid  or  alkaline  alcohol 
can  be  made  to  give  the  staining  effect  desired,  is  that  of  Hospital 
Steward  R.  W.  King,  U.  S.  Navy.  The  preparation  of  the  stain  is  as 
follows : 

Dissolve  i  gram  of  Grubler's  methylene  blue  and  0.5  gram  of 
sodium,  bicarbonate  in  100  c.c.  of  distilled  water.  Transfer  to  porce- 
lain dish  and  evaportate  to  dryness  over  Bunsen  burner  or  alcohol 
flame.  The  fluid  may  be  allowed  to  boil  gently  until  about  half  of  the 
water  has  escaped,  when  the  heat  should  be  somewhat  reduced. 
Evaporation  is  now  facilitated  by  causing  the  fluid  to  flow  upon  the 
sides  of  the  dish  by  tilting.  This  also  overcomes  the  tendency  to 
spluttering.  The  heat  should  be  continued  until  the  last  trace  of 
moisture  has  disappeared.  The  absolutely  dry  stain  is  then  removed 
from  the  dish  and  preserved  in  small  well-stoppered  vials. 

(If  larger  quantities  than  the  above  are  to  be  made,  separate  dishes 
must  be  used.) 

Stock  solution:  Dissolve  0.3  gram  of  the  polychromatic  blue  and 
0.175  gram  of  Grubler's  eosin  in  100  c.c.  of  pure  methyl  alcohol. 
Allow  to  stand  for  three  hours  and  filter.  Add  25  drops  of  3%  hydro- 
chloric acid  alcohol  (used  in  T.  B.  staining). 

For  use,  take  25  c.c.  of  the  stock  solution  and  add  eight  drops  of  the 
alkaline  alcohol  (5  c.c.  10%  sol.  caustic  soda  to  100  c.c.  95%  alcohol), 
and  test  by  staining  a  section  of  a  fresh  blood  film.  If  the  blue  over- 
stains,  add  one  drop  of  the  acid  alcohol  and  test  again.  If  the  eosin 
overstains  (nuclei  stain  poorly),  add  two  or  three  drops  of  the  alkaline 
alcohol.  In  this  way  it  will  only  require  one  or  two  trials  to  adjust 
the  staining  properties  of  the  fluid,  after  which  it  will  keep  unchanged 
for  some  time.  At  any  time,  however,  if  it  is  found  not  to  give  good 


ROMANOWSKY   STAINING    METHODS.  151 

results,  the  addition  of  a  drop  or  so  of  either  the  acid  or  alkaline 
alcohol,  as  indicated,  will  restore  its  original  staining  properties.  The 
acid  and  alkaline  alcohols  may  be  used  in  the  same  way  and  with 
about  the  same  results  with  Wright's  staining  fluid. 

In  all  Romanowsky  methods  distilled  water  should  be  used.  If  not 
obtainable,  the  best  substitute  is  rain-water  collected  in  the  open  and 
not  from  a  roof. 

Method  of  staining: 

1.  Make  films  and  air  dry. 

2.  Cover  dry  film  preparation  with  the  methyl-alcohol  stain  for  i 
minute  (to  fix). 

3.  Add  water  to  the  stain  on  the  cover-glass  or  slide,  drop  by  drop, 
until  a  yellow  metallic  scum  begins  to  form.     It  is  advisable  to  add  the 
drops  of  water  rapidly  in  order  to  eliminate  precipitates  on  the  stained 
film.     Practically,  we  may  add  i  drop  of  water  for  every  drop  of  stain 
used. 

4.  Wash  thoroughly  in  water  until  the  film  has  a  pinkish  tint. 

5.  Dry  with  filter-paper  and  mount. 

Red  cells  are  stained  orange  to  pink;  nuclei  shades  of  violet; 
eosinophile  granules,  red;  neutrophile  granules,  yellow  to  lilac;  blood 
platelets,  purplish;  malarial  parasites,  blue;  chromatin,  metallic  red  to 
rose-pink. 

Giemsa's  Modification  of  the  Romanowsky  Method. — This  is  one  of  the 
most  perfect  of  the  modifications.  The  objection  is  that  greater  time 
in  staining  films  is  required  than  with  the  Wright  or  Leishman  method 
and  the  stain  is  very  expensive. 

Take  of  Azur  II  eosin  0.3  gm.     Azur  II  0.08  gm. 

Dissolve  this  amount  of  dry  powder  in  25  c.c.  of  glycerin  at  60°  C. 
Then  add  25  c.c.  of  methyl  alcohol  at  the  same  temperature.  Allow 
the  glycerin  methyl- alcohol  solution  to  stand  over  night  and  then 
filter.  This  is  the  stock  stain.  To  use:  Dilute  i  c.c.  with  10  to  15  c.c. 
of  water.  If  i  to  1000  potassium  carbonate  solution  is  used  instead  of 
water  it  stains  more  deeply.  Having  fixed  the  smear  with  methyl 
alcohol  for  5  minutes,  pour  on  the  diluted  stain  and  after  15  to  30  min. 
wash  off  and  continue  washing  with  distilled  water  until  the  film  has 
a  slight  pink  tinge.  For  Treponema  pallidum  stain  from  2  to  12  hours. 


I$2  MICROMETRY    AND    BLOOD    PREPARATIONS. 

While  the  Romanowsky  methods  are  more  satisfactory  for  differential 
counts  and  for  the  demonstration  of  the  malarial  parasites,  and  es- 
pecially for  differentiating  species,  yet  by  reason  of  the  liability  to 
deterioration  in  the  tropics  of  methylene  blue  the  haematoxylin  methods 
may  be  preferable.  Many  workers  in  blood-work  and  cytodiagnosis 
prefer  the  haematoxylin. 

1.  Fix  the  film  either  by  heat  or  with  Whitney's  fixative. 
Heat  is  to  be  preferred. 

2.  Stain  with  Meyer's  hemalum  or  Delafield's  haematoxylin 
for  from  5  to  15  minutes  according  to  the  stain.     Fre- 
quently 3  minutes  will  be  found  sufficient.     To  make  the 
hemalum,  dissolve  .5  gm.  of  haematin  in  25  c.c.  of  95% 
alcohol.     Next  dissolve  25  gm.  of  ammonia  alum  in  500 
c.c.  of  distilled  water.     Mix  the  two  solutions  and  allow  to 
ripen  for  a  few  days.     The  stain  should  be  satisfactory  in 
2  or  3  days. 

To  make  Delafield's  haematoxylin,  dissolve  i  gm.  of 
haematoxylin  crystals  in  6  c.c.  of  95%  alcohol.  Add  this 
to  100  c.c.  of  saturated  aqueous  solution  of  ammonia  alum. 
After  exposure  to  light  for  a  week,  the  color  changes  to  a 
deep  blue-purple.  Add  to  this  ripened  stain  25  c.c.  of 
glycerin  and  25  c.c.  of  methyl  alcohol  and,  after  it  has 
stood  for  about  two  days,  filter.  The  stain  should  be 
filtered  from  time  to  time  as  a  sediment  forms.  This 
makes  a  stock  solution  which  should  be  diluted  10  to  15 
times  with  water  when  staining. 

3.  Wash  for  2  to  5  minutes  in   tap  water   to  develop   the 
haematoxylin  color. 

4.  Stain  either  with  a  i  to  1000  aqueous  solution  of  eosin  or 
with  a  1/2  of  i%  eosin  solution  in  70%  alcohol.     The 
eosin  staining  only  requires  15  to  30  seconds. 

5.  Wash  and  examine. 

IODOPHILIA. 

This  reaction  is  supposed  to  be  due  to  the  presence  of  glycogen, 
especially  in  the  polymorphonuclears,  in  suppurative  conditions. 


IODOPHILIA   REACTION.  153 

Make  blood-smears  on  cover-glasses  as  usual,  and  after  they  dry, 
but  without  fixation,  mount  them  in  a  drop  of  the  following  solution: 

Iodine,  i  part. 

Potassium  iodide,     3  parts. 
Gum  arabic,  50  parts. 

Water,  100  parts. 

Small  brown  masses  in  the  polymorphonuclears  or  lying  extra- 
cellular indicate  a  positive  iodophilia. 


CHAPTER  XIV. 
NORMAL  AND  PATHOLOGICAL  BLOOD. 

In  considering  what  may  be  termed  normal  blood,  it  must  be  borne 
in  mind  that  the  normal  varies  for  men,  women  and  children: 

Hb.  Red  cells.         Leukocytes. 

Men,         90  to  110%,  5  to  5  1/2  million,     7500. 

Women,    80  to  100%,  4  1/2  to  5  million,     7500. 

Children,  70  to  80%,  4  1/2  to  5  million,     9000. 

COLOR-INDEX. 

This  is  obtained  by  dividing  the  percentage  of  the  haemoglobin  by 
the  percentage  of  red  cells,  five  million  red  cells  being  considered  as 
100%.  To  obtain  the  percentage  of  red  cells  it  is  only  necessary  to 
multiply  the  two  extreme  figures  to  the  left  by  two.  Thus  if  a  count 
showed  the  presence  of  1,700,000  red  cells,  the  percentage  would  be 
34.  (17x2  =  34.)  If  the  Hb.  percentage  in  this  case  were  50;  then 
the  color  index  would  be  50  -H  34,  or  1.4. 

In  normal  blood  the  color-index  is,  approximately,  i. 

In  anaemias  we  have  three  types  of  color-index:  (i)  The  perni- 
cious anaemia  type,  which  is  above  i.  Here  we  have  a  greater  re- 
duction in  red  cells  than  we  have  of  the  haemoglobin  content  of  each 
cell.  (2)  The  normal  type,  when  both  red  cells  and  haemoglobin  are 
proportionally  decreased,  as  in  anaemia  following  haemorrhage.  (3) 
The  chlorotic  type.  Here  there  is  a  great  decrease  in  haemoglobin 
percentage,  but  only  a  moderate  decrease  in  the  number  of  red  cells. 
Hence  the  color-index  is  only  a  fraction  of  i.  For  example,  in  a  case  of 
chlorosis  we  have  40%  of  haemoglobin  and  90%  of  red  cells,  40  -j-  90  =  .4 

RED  CELLS. 

In  considering  the  corpuscular  richness  of  a  specimen  of  blood,  it 
must  be  remembered  that  this  does  not  necessarily  bear  any  relation  to 

154 


NUCLEATED    RED    CELLS.  155 

the  quantity  of  blood  in  the  body.  Thus,  a  more  or  less  bloodless- 
looking  individual,  the  total  quantity  of  whose  blood  is  greatly  reduced, 
may  notwithstanding  give  a  normal  red  count.  In  examining  a 
specimen  of  peripheral  blood  we  get  a  qualitative,  not  a  quantitative 
result. 

Normally,  we  have  an  increase  in  red  cells  in  those  living  at  high 
altitudes.  An  altitude  of  two  thousand  feet  increases  the  red  count 
about  one  million,  and  a  height  of  six  thousand  feet  about  two  million. 
Profuse  sweats  and  diarrhoeas  also  increase  the  red  count.  Pathologi- 
cally, in  chronic  polycythemia  with  cyanosis  and  splenic  enlargement, 
we  have  a  red  count  of  about  ten  million.  In  cyanosis  from  heart 
disease,  etc.,  and  in  Addison's  disease  there  is  also  an  increase  in  red 
cells. 

The  normal  red  cell  or  erythrocyte  measures  about  7.5/4  in  diameter. 
It  is  nonnucleated  and  normally  stains  with  acid  dyes,  taking  the  pink 
of  eosin  or  the  orange  of  orange  G.  If  larger,  10  to  20^,  it  is  called  a 
macrocyte;  if  smaller,  3  to  6/j,  a  microcyte. 

Macrocytes  are  rather  indicative  of  severe  forms  of  anaemia,  the 
microcytes,  of  less  grave  types.  When  the  red  cell  is  distorted  in  shape, 
it  is  called  a  poikilocyte.  Care  must  be  exercised  that  distorted  shapes 
are  not  due  to  faulty  technic.  Crenation  and  vacuolation  of  red  cells 
are  marked  in  poorly  prepared  specimens. 

In  addition  to  variation  in  size  and  shape,  we  also  have  pathological 
variation  in  staining  affinities. 

Poly chromatophilia.— This  shows  itself  by  red  cells  taking  a 
brownish  to  a  dirty  blue  tint,  as  is  frequently  seen  in  immature  red  cells, 
especially  nucleated  ones. 

Granular  basophilic  degeneration  (also  termed  punctate  baso- 
philia  and  stippling)  refers  to  the  presence  of  blue  dots  in  the  pink 
back-ground  of  stained  red  cells.  It  is  found  in  many  severe  anaemias, 
as  pernicious  anaemia,  the  leukaemias,  malarial  cachexia,  etc.  It  is 
very  characteristic  of  lead  poisoning. 

The  nucleated  red  cell,  while  normal  for  the  marrow,  is  always 
pathological  for  the  blood  of  the  peripheral  circulation.  Normoblasts 
have  the  diameter  of  a  normal  red  cell.  The  nucleus  is  round  and 
stains  intensely  with  basic  dyes,  often  appearing  almost  black. 


156  NORMAL    AND    PATHOLOGICAL    BLOOD. 

Another  characteristic  is  that  it  frequently  appears  as  does  the  setting 
in  a  ring.  Some  give  the  term  microblast  to  smaller  nucleated  forms. 
In  normoblasts  the  red  cell  proper  stains  normally.  The  megaloblasts 
not  only  have  a  greater  diameter  than  the  normoblast,  but  the  nucleus 
is  poor  in  chromatin,  stains  less  intensely  and  is  less  distinctly  out- 
lined. Instead  of  being  round,  the  nucleus  is  irregular  and  may  be 
trefoil  in  shape.  The  cytoplasm  surrounding  the  nucleus  shows 
polychromatophilia.  This  contrasted  with  the  pure  blue  of  the 
lymphocytes  should  differentiate.  Normoblasts  are  found  in  secondary 
anaemias,  and  especially  in  myelogenous  leukaemia.  Megaloblasts 
are  peculiarly  characteristic  of  pernicious  anaemia.  Enormous 
megaloblasts  are  sometimes  termed  gigantoblasts. 

In  aplastic  anaemia  (a  severe  type  of  pernicious  anaemia),  in  con- 
trast to  ordinary  pernicious  anaemia,  nucleated  reds  are  very  rarely 
found.  There  is  also  very  little  poikilocytosis,  and  the  color-index  is 
about  normal.  It  is  a  rare,  rapidly  fatal  anaemia,  particularly  of 
young  women. 

WHITE  CELLS. 

Owing  to  the  conflicting  views  as  to  origin,  nature  and  functions  of 
the  various  leukocytes,  their  classification  is  in  a  state  of  confusion. 
As  regards  the  appearance  of  the  cells,  this  of  course  varies  as  "the  stain 
used,  and  it  requires  considerable  experience  for  a  single  individual  to 
be  able  to  positively  recognize  the  difference  between  a  lymphocyte  and 
a  large  mononuclear  when  one  specimen  is  stained  with  a  Roman- 
owsky  stain,  another  with  Ehrlich's  triacid,  and  a  third  with  haema- 
toxylin  and  eosin.  This,  of  course,  is  intensified  when  different  persons 
adhere  to  the  method  of  staining  which  they  prefer  and  are  at  a  loss  to 
appreciate  differences  which  are  brought  out  by  some  other  stain  used  by 
some  other  person.  Even  with  the  same  stain  used  with  different  speci- 
mens of  blood  we  find  the  staining  characteristics  of  various  leukocytes 
imperceptibly  merging,  the  one  into  the  other,  so  that  at  times  it  is 
impossible  for  one,  even  with  his  own  standard  of  differentiation,  to  be 
sure  whether  he  is  dealing  with  a  lymphocyte  or  a  large  mononuclear. 
The  difficulty  is  even  greater  when  wre  deal  with  Turck's  irritation 
forms  and  with  myelocytes. 


LYMPHOCYTES.  157 

Without  going  into  the  various  granule  stainings  so  thoroughly 
brought  out  by  Ehrlich,  we  shall  immediately  take  up  the  question  of 
a  practical  classification  for  use  in  making  a  differential  count.  As  the 
Romanowsky  method  of  staining  (Wright,  Leishman  or  Giemsa) 
gives  us  information  not  yielded  by  either  haematoxylin  and  eosin  or 
the  triacid,  the  points  of  differentiation  to  be  referred  to  in  that  which 
follows  is  with  blood  so  stained. 

In  considering  the  staining  affinities  of  different  parts  of  the 
leukocytes,  it  is  convenient  to  divide  such  into  basic  ones,  acid  ones  and 
those  which  may  be  said  to  be  on  the  border  line  betwreen  these — the 
so-called  neutrophilic  affinities. 

With  Wright's  stain  we  have  the  eosinophile  cr  oxyphile  affinity  of 
the  granules  of  eosinophiles  for  acid  dyes,  in  this  case  eosin.  The 
nuclei  and  basophile  granules  have  affinities  in  greater  or  less  degree 
for  basic  stains  (the  blue  and  the  violet  shading  resulting  from  methylene 
blue  as  modified  by  polychroming).  With  the  granules  in  the  cyto- 
plasm of  the  polymorphonuclears  and  neutrophilic  myelocytes,  and  to  a 
less  extent  in  the  transitional,  we  have  a  staining  which  merges  into 
a  yellowish-red  on  the  one  extreme  and  into  a  lilac  on  the  other. 
As  a  standard,  neutrophilic  granules  should  be  a  mean  of  these 
extremes. 

Not  only  by  reason  of  the  authority  of  Ehrlich,  but  because  such  a 
division  gives  all  variations,  which  can  then  be  combined  by  one 
preferring  a  simpler  classification,  it  would  seem  proper  to  divide  the 
normal  leukocytes  into: 

1.  Small  lymphocytes.     These  are  small  round  cells  about  the 
size  of  a  red  corpuscle  with  a  large  centrally-placed,  deeply  violet  stain- 
ing nucleus  and  a  narrow  zone  of  cytoplasm.     This  cytoplasm  may 
not  be  more  than  a  mere  crescentic  fringe.     This  is  the  type  of  lymph- 
ocytes which  makes  up  the  greater  proportion  of  the   leukocytes  in 
chronic  lymphatic  leukaemia.      At  times  these  cells  seem  to  be  com- 
posed of  nucleus  alone. 

2.  Large   lymphocytes.     These  are  of  the  same  type  as  small 
lymphocytes,  but  possessing  more  cytoplasm.  The  nucleus,  while  round 
and  taking  a  fairly  deep  rich  violet  stain,  does  not  stain  so  deeply  as  the 
nucleus  of  the  small  lymphocytes.     The  cytoplasm  is  a  clear  translucent 


158  NORMAL   AND    PATHOLOGICAL   BLOOD. 

pure  blue.  It  may  contain  pinkish  granules  known  as  azur  granules, 
but  these  are  of  rather  large  size  and  do  not  mar  the  glass-like 
appearance.  They  are  from  9  to  15/1  in  diameter  and  are  com- 
mon in  children.  In  the  acute  lymphatic  leukaemias  they  at  times 
predominate. 

3  Large  mononuclears.  These  are  large  round  or  oval  cells  with 
a  nucleus  which  has  lost  the  richness  of  violet  staining  of  the  lympho- 
cyte nucleus.  The  nucleus  is  furthermore  frequently  irregular  in  outline 
or  may  show  the  commencing  indentation  of  the  transitional  nucleus. 

There  is  not  that  sharp  distinction  between  nucleus  and  cytoplasm 
that  exists  in  the  lymphocytes.  The  cytoplasm  of  the  large  mononu- 
clear  gives  the  impression  of  opacity,  as  if  it  were  frosted  glass  instead 
of  clear  glass.  The  neutrophile  mottling  which  begins  to  appear 
causes  a  disappearance  of  the  pure  blue  character  of  the  cytoplasm 
of  the  lymphocyte.  It  is  principally  by  the  washed-out  staining 
of  the  nucleus  and  the  opaque  lilac  of  the  cytoplasm,  that  we 
differentiate  them  from  the  lymphocytes.  They  greatly  resemble 
Turck's  irritation  forms  or  plasma  cells  and  may  be  confused  with 
myelocytes. 

4.  Transitionals.  These  appear  as  but  a  later  stage  in  the 
decay  of  the  large  mononuclears;  the  nucleus  is  more  indented, 
frequently  horse-shoe  shaped,  and  has  a  washed  out  violet  shade  of 
less  intensity  than  that  of  the  large  mononuclears.  These  are  the 
cells  so  often  disrupted  in  smears. 

These  four  kinds  of  cells  are  frequently  referred  to  as  the  lymphocyte 
series,  and  although  many  authorities  consider  that  the  small  lympho- 
cyte represents  a  more  mature  cell  than  the  others  of  this  class,  yet  it 
is  thought  by  others  that  the  age  of  the  cell  increases  as  we  go  from 
small  lymphocytes  to  large  lymphocytes,  thence  to  the  large  mononu- 
clear;  and  then  in  the  transitional  we  have  the  decrepit  stage  which 
precedes  dissolution.  The  old  view  that  the  transitional  was  the 
precursor  of  the  polymorphonuclear  has  few  advocates  at  the  present 
time. 

While  it  is  convenient  to  consider. these  hyaline  cells  as  representing 
different  stages  in  development,  yet  from  a  stand-point  of  immunity 
this  is  untenable.  The  large  mononuclears  and  transitionals  are  the 


GRANULAR    LEUKOCYTES.  159 

cells  in  which  we  find  certain  animal  cells  and  pigment  phagocytized,  as 
is  the  case  in  malaria.  These  cells  are  the  macrophages  of  Metch- 
nikoff  and  are  probably  derived  from  the  bone  marrow. 

The  lymphocytes  take  origin  from  the  lymphoid  tissue,  and  very 
probably  the  large  lymphocyte  is  a  younger,  more  immature  cell  than 
the  small  lymphocyte.  Consequently,  we  probably  have  marrow 
lymphocytes  and  gland  lymphocytes. 

In  addition  to  the  series  of  leukocytes  just  considered  we  have 
present  normally  in  the  blood  three  types  of  granular  cells  distinguished 
according  to  the  staining  affinity  of  their  granules.  These  are : 

1 .  Polymorphonuclear  leukocytes.    This  cell  normally  constitutes 
the  greater  proportion  of  the  leukocytes.     It  is  an  amoeboid,  actively 
phagocytic  cell,  about  10  or  i2/x  in  diameter,  and  is  the  microphage  of 
Metchnikoff.     Bacteria  are  actively  phagocytized  by  this  cell,  and  it  is 
the  cell  concerned  in  determining  the  opsonic  power  of  blood  to  various 
bacteria.     It  has  fine  lilac  granules  which  are  termed  neutrophilic. 
(epsilon  granules).     The  single  nucleus  is  rich  in  character  and  is 
lobose  like  the  kernel  of  an  English  walnut;  frequently  it  resembles  the 
letter  z.     These  cells  are  derived  from  the  neutrophilic  myelocytes  of 
the  bone  marrow.     It  is  in  these  cells  that  the  glycogen,  or  iodophil, 
granules  appear  in  certain  suppurative  conditions. 

2.  Eosinophile  Leukocytes. — These  are  very  striking  cells  with 
coarse  granules  staining  brilliantly  pink,  the  eosinophile,  oxyphile  or 
acidophile  granules  (alpha  granules  of  Ehrlich).     The  cells  are  a  little 
larger  than  the  polymorphonuclears.     The  normal  eosinophile  is  to  be 
distinguished  from  the  eosinophilic  myelocyte  by  its  possessing  two 
distinct  lobes  in  the  nucleus.     The  nucleus  of  the  myelocyte  is  round. 
The  eosinophile  is  the  cell  so  frequently  increased  in  infections  by 
intestinal  animal  parasites. 

3.  Mast  Cells. — These  also  have  coarse  granules,  but  they  stain 
a   deep  violet  blue.      Hence  they  are  basophile  granules   (gamma 
granules).      In    fresh    blood   these  granules  do  not  show  up   very 
well,    thus   they   can    be    distinguished    from    the    highly    refractile 
granules  of  the  eosinophile.     The-tri-lobed  nucleus  stains  less  intensely 
than  the  granules.     As  a  rule,  the  mast  cell  is  about  the  size  of  a 
pol\  morphonuclear. 


l6o  NORMAL   AND    PATHOLOGICAL   BLOOD. 

In  a  differential  count  of  normal  blood  we  find  about  the  following 
percentages. 

Polymorphonuclears,  65  to  70%,  About  5000  per  c.  mm. 
Small  lymphocytes,  20  to  25%,  About  1500  per  c.  mm. 
Large  lymphocytes,  5  to  10%,  About  500  per  c.  mm. 
Large  mononuclears,  i  to  2%,  About  100  per  c.  mm. 
Transitionals,  2  to  4%,  About  200  per  c.  mm. 

Eosinophiles,  i  to    2%,  About  100  per  c.  mm. 

Mast  cells,  1/4  to  1/2%,  About      25  per  c.  mm. 

The  leukocytes  which  are  found  in  the  peripheral  circulation  only 
in  pathological  conditions  are: 

1.  Neutrophilic  Myelocytes. — The  common  type  is  a  large  cell 
with  a  large  centrally-placed,  feebly-staining  nucleus.     This  may  be 
recognized  by  the  difficulty  of  distinguishing  the  nucleus  from  the 
cytoplasm,  there  being  no  sharp  line  separating  these  parts  of  the  cell. 
They  imperceptibly  merge  into  one  another.     They  differ  from  a 
large  mononuclear  in  that  the  cytoplasm  is  distinctly  dotted  with 
neutrophile  granules,  and  that  we  cannot  make  out  a  distinct  line  of 
separation  of  a  slightly  irregular  or  indented  nucleus  from  the  sur- 
rounding slightly  neutrophilic  cytoplasm.     Cornil  has  described  a  very 
large  myelocyte  with    eccentrically-placed  nucleus  and  neutrophilic 
granules. 

Myelocytes  are  at  times  found  with  both  basophilic  and  neutrophilic 
granules,  and  may  rarely  be  seen  to  have  all  three  kinds  of  granules 
on  a  single  myelocyte,  acidophile,  basophile  and  neutrophile. 

2.  Eosinophilic  Myelocytes. — These  can  be  distinguished  from 
normal  eosinophiles  by  their  possessing  a  single  round  nucleus,   not 
bilobed.     These  myelocytes  may  be  as  large  as  a  normal  eosinophile, 
but  frequently  are  no  larger  than  a  red  cell. 

The  neutrophile  myelocyte  is  characteristic  of  spleno-myelogenous 
leuka&mia,  the  eosinophile  one  of  myelogenic  leukaemia.  The  oc- 
currence of  an  occasional  myelocyte  is  frequently  noted  in  conditions 
having  a  Jeukocytosis.  In  diphtheria  their  presence  in  numbers  is  of 
bad  prognostic  import.  Myelocytes  are  of  diagnostic  importance  in 
metastases  of  malignant  tumors. 


BLOOD    PLATELETS  AND    HEMOKONIA.  l6l 

3.  The  Irritation  Cell  of  Turck,  or  Plasma  Cell.— This  cell  has  a 
faintly-staining,  eccentrically-placed  nucleus,  and  a  dark  opaque  blue, 
frequently  vacuolated,  cytoplasm.  They  are  usually  recorded  as  large 
mononuclears. 

BLOOD  PLATELETS. 

These  are  normally  present  in  blood  in  the  number  of  about  350,000 
per  cubic  millimeter.  They  disintegrate  very  quickly  after  the  blood  is 
withdrawn.  Wright  has  demonstrated  that  they  are  pinched-off 
projections  of  giant  cells  of  the  bone  marrow.  They  consist  only  of 
protoplasm,  no  nuclear  material.  They  do  not  contain  haemoglobin. 
In  conditions  where  giant  cells  are  less  abundant,  as  in  pernicious 
anaemia,  the  blood  platelets  are  less  abundant.  In  myelogenous 
leukemia  they  are  very  abundant.  They  vary  in  size  from  2  to  5/1 
according  as  a  larger  or  smaller  pseudopod  of  a  giant  cell  has  been 
broken  off.  Stained  with  Wright's  stain,  they  are  more  purplish  than 
blue  and  show  thread-light  projections.  They  are  often  mistaken  for 
the  protozoal  causes  of  various  diseases.  Especially  are  they  confused 
with  malarial  parasites  when  lying  on  a  red  cell.  The  blood  plate  has 
no  brick-red  chromatic  material;  it  is  purplish  rather  than  blue,  and 
has  no  pigment  grains.  It  is  advisable  to  compare  these  isolated  blood- 
plates  writh  the  larger  or  smaller  aggregations  scattered  about  the 
smears.  In  this  way  their  true  character  is  apparent.  In  addition  to 
blood  platelets,  which  in  fresh  blood  can  only  be  observed  when  a 
fixative  is  used,  we  have  other  confusing  bodies.  The  hemokonia  of 
Muller  are  small,  highly  refractile  bodies  showing  active  oscillatory 
movement.  They  are  supposed  to  be  cast-off  granules  of  eosinophiles 
or  other  leukocytes.  Pinched  off  fragments  of  red  cells  may  also 
appear  as  possible  protozoal  bodies. 

LEUKOPENIA. 

This  is  a  term  used  to  designate  a  reduction  in  the  normal  number 
of  leukocytes.  A  leukocyte  count  of  5000  would  represent  a  slight 
leukopenia;  one  of  2000,  a  marked  leukopenia.  In  the  later  stages  of 
typhoid,  and  in  acute  miliary  tuberculosis,  we  expect  a  moderate 
leukopenia.  Chronic  alcoholism  and  chronic  arsenic  poisoning  cause 


162  NORMAL    AND    PATHOLOGICAL    BLOOD 

a  reduction  in  the  number  of  the  white  cells.  Pernicious  anaemia 
shows  a  marked  leukopenia,  as  is  also  the  case  with  Banti's  disease. 
Two  tropical  diseases,  Kala-azar  and  dengue,  show  a  marked  leuko- 
penia, the  counts  often  being  below  2500.  During  the  apyrexial 
period  of  malaria  we  may  have  a  white  count  of  5000. 

EOSINOPHILIA. 

Where  the  eosinophiles  are  increased  to  5%,  we  have  a  moderate 
eosinophilia.  In  some  cases  of  infection  with  intestinal  parasites, 
especially  hook-worms,  but  also  from  other  parasites,  as  round  and 
whip-worms,  we  may  have  an  eosinophilia  of  30  to  50%.  In  Guam, 
among  the  natives,  it  is  difficult  to  find  an  eosinophile  count  under  15%. 

The  eosinophilia  of  trichinosis  is  best  known,  and  a  combination  of 
this  blood  finding  with  fever  and  marked  pains  of  muscles,  would 
justify  the  excision  of  a  piece  of  muscle  for  examination  for  encysted 
embryos.  In  true  asthma  eosinophilia  is  marked,  and  its  absence  is  of 
value  in  indicating  other  causes  for  the  condition.  Certain  skin 
diseases,  especially  pemphigus,  also  show  eosinophilia. 

LEUKOCYTOSIS. 

It  is  to  an  increase  in  the  polymorphonuclears  that  this  term  is 
usually  applied,  the  term  lymphocytosis  or  eosinophilia  being  em- 
ployed where  white  cells  of  eosinophile  or  lymphocyte  nature  are 
increased.  We  have  physiological  leukocytosis  in  the  latter  weeks  of 
pregnancy,  also  in  the  new-born,  and  in  connection  with  digestion. 

Pathological  Leukocytosis. — Pneumonia.  In  this  disease  we 
have  a  leukocytosis  of  20,000  to  30,000  or  higher.  The  eosinophiles 
are  almost  absent.  A  normal  leukocyte  count  in  pneumonia  makes  a 
prognosis  unfavorable. 

Septic  processes.  The  leukocyte  count  is  of  great  value,  especially 
when  we  obtain  a  leukocytosis  with  80  to  90%  of  polymorphonuclears, 
as  in  appendicitis,  cholecystitis  or  other  suppurative  conditions. 

According  to   Cabot,  leukocytosis  varies  in  infections  as  follows: 

i.  Severe    infection — good    resistance;    early    marked    and 
persistent  leukocytosis. 


LEUKOCYTOSIS.  163 

2.  Slight  infection — slight  resistance;  leukocytosis  present, 

but  not  marked. 

3.  In   fulminating   infections  \ve    may  have   no   increase  in 

whites,  but  a  higher  percentage  of  polymorphonuclears. 

4.  Slight   infection   and   good   resistance    may  not  be  pro- 

ductive of  leukocytosis. 

Spirochaeta  fevers,  as  relapsing  fever,  may  give  a  leukocytosis  of 
from  25,000  to  50,000. 

Small-pox,  especially  at  time  of  pustulation,  plague,  scarlet  fever 
and  liver  abscess  give  a  leukocytosis  of  from  12,000  to  15,000. 


FIG.  52. — Leukocytosis  (40,000);  sixteen  polymorphonuclears  in  field.     (Cabot.) 

Erysipelas  and  epidemic  cerebrospinal  meningitis  also  give  a 
leukocytosis  of  from  15,000  to  20,000.  In  malignant  diseases  we  some- 
times have  a  moderate  leukocytosis.  Rogers  states  that  in  liver  abscess, 
with  a  leukocytosis  of  15,000  to  20,000,  we  have  onlyabout  75  to  77% 
of  polymorphonuclears — there  being  also  a  moderate  increase  in  the 
percentage  of  large  mononuclears. 

LYMPHOCYTOSIS. 

Of  course,  the  disease  in  which  we  have  the  most  marked  lymphocy- 
tosis  is  lymphatic  leukaemia. 

Whooping-cough  may  give  a  lymphocytosis  of  20,000  to  30,000. 


164  NORMAL    AND    PATHOLOGICAL    BLOOD. 

Young  children  have  normally  an  excessive  proportion  of  lympho- 
cytes. This  is  apt  to  be  particularly  marked  in  hereditary  syphilis. 
Enlarged  tonsils  may  give  rise  to  a  lymphocytosis  of  10,000  to  15,000, 
when  more  than  50%  of  the  white  cells  will  be  lymphocytes.  Rickets 
and  scurvy  give  a  lymphocytosis. 

DISEASES  IN  WHICH  THERE  is  A  NORMAL  LEUKOCYTE  COUNT. 

Uncomplicated  tuberculosis,  influenza,  Malta  fever,  measles, 
trypanosomiasis,  malaria,  syphilis  and  chlorosis.  In  malaria  we  have 
a  leukocytosis  at  the  time  of  the  rigor,  while  during  the  apyrexial 
period  there  is  a  moderate  leukopenia.  In  malaria  we  have  a  marked 
increase  in  the  percentage  of  the  large  mononuclears  and  transitionals. 
These  may  form  from  25%  to  35%  of  the  leukocytes.  When  bearing 
particles  of  pigment  they  are  known  as  melaniferous  leukocytes — 
macrophages  which  have  ingested  malarial  material.  In  dengue,  at 
the  time  of  the  terminal  rash,  we  may  have  as  great  a  percentage  of 
large  mononuclears.  In  this  disease,  however,  we  have  a  great  dimi- 
nution of  polymorphonuclears  from  the  start  (25  to  40%).  Instead  of 
a  large  mononuclear  we  have  at  the  onset  a  lymphocytic  increase. 
There  is  an  increase  of  large  mononuclears  in  trypanosomiasis. 

THE  PRIMARY  ANEMIAS. 

Chlorosis. — In  chlorosis  it  is  the  reduction  of  haemoglobin  with  the 
slight  numerical  variation  from  normal  of  the  red  cells  that  makes  for 
a  diagnosis.  The  color-index  is  very  low.  There  is  nothing  abnormal 
about  the  leukocytes.  Microcytes  may  be  present,  and  very  occasion- 
ally a  normoblast.  Macrocytes  and  megaloblasts  are  always  absent. 
Blood  of  chlorotics  is  very  pale  and  very  fluid  and  coagulates  rapidly, 
hence  frequency  of  thrombosis. 

Simple  Primary  Anaemia. — This  condition  is  not  recognized  by 
many  authors,  but  is  a  convenient  term  under  which  to  group  anaemias 
which  are  neither  chlorosis  nor  pernicious  anaemia  and  for  which  no 
assignable  cause  can  be  designated.  It  is  a  secondary  anaemia  with- 
out a  cause.  In  it  color-index  is  about  normal,  there  is  no  change  in 
the  leukocytes  and  cases  go  on  to  recovery. 

Pernicious  Anaemia. — In  pernicious  anaemia  we  obtain  a  very 


PERNICIOUS  ANAEMIA.  165 

fluid,  but  normally-colored  drop  of  blood  upon  puncture.  The  yellow 
marrow  of  the  long  bones  is  transformed  into  a  soft,  bright  red  lymphoid 
tissue,  smears  from  which  show  great  numbers  of  megaloblasts. 
Areas  of  fatty  degeneration  are  characteristic,  especially  the  tiger-lily 
spots  in  the  heart  muscle.  Iron-containing  pigment  (hemosiderin)  is 
found  in  the  liver,  spleen  and  kidneys.  Areas  of  degeneration  in  the 
spinal  cord  may  account  for  nervous  symptoms.  The  red  cells  fre- 
quently fall  below  2,000,000  with  patients  going  about.  Cases  have 
been  reported  with  counts  under  200,000.  The  color-index  is  high. 
Megaloblasts  are  the  most  characteristic  qualitative  change  in  the  red 


FIG.  53. — Pernicious  anaemia.     M.m,  Megaloblasts;  n,  normoblast; 
s,  stippling  (punctate  basophilia).     (Cabot.) 

cells.  Megaloblastic  crises  may  at  certain  times  show  enormous 
numbers  of 'megaloblasts.  Cases  often  present  remissions  in  which  no 
megaloblasts  can  be  found.  In  such  cases  the  presence  of  many 
macrocytes  should  prevent  an  examiner  reporting  against  a  pernicious 
anaemia  previously  diagnosed. 

Poikilocytosis,  polychromatophilia  and  stippling  are  also  features  of 
the  disease.  Normoblasts  are  far  less  frequent  than  megaloblasts  and 
there  is  usually  a  moderate  lymphocytosis.  Myelocytes  may  be 
present.  Cases  of  pernicious  anaemia  show  remissions  during  which 
the  patient  is  apparently  on  the  road  to  recovery.  Such  improvements 
are  only  temporary.  The  remissions  may  last  from  two  months  to 


1 66  NORMAL   AND    PATHOLOGICAL   BLOOD. 

possibly  three  or  four  years.  Especially  in  the  anaemia  of  Dibothrio- 
cephalus  latus  do  we  have  a  picture  of  pernicious  anaemia.  It  is 
supposed  to  be  due  to  a  toxin  present  in  the  heads  of  these  tape- worms. 

\ 
SECONDARY  ANEMIAS. 

These  are  the  anaemias  which  can  be  definitely  traced  to  some 
disease  not  of  the  haemopoietic  system.  In  some  secondary  anaemias, 
as  in  syphilis,  carcinoma  and  tuberculosis,  we  have  a  chlorotic  color- 
index  (chloro  anaemias). 

In  secondary  anaemias  polychromatophilia,  poikilocytosis  and 
punctate  basophilia  (stippling)  may  be  present.  This  latter  is  very 
marked  in  lead  poisoning,  but  in  certain  cases  of  malarial  cachexia  it 
may  be  equally  prominent.  The  only  form  of  nucleated  red  cell  seen  is 
the  normoblast,  in  very  small  numbers,  or  it  may  not  be  present, 

Megaloblasts  are  practically  never  seen,  except  in  some  of  the  very 
severe  parasitic  anaemias,  as  the  broad  Russian  tape-worm  infection. 
The  red  cells  generally  number  between  2,000,000  and  4,000,000,  thus 
differentiating  chlorosis.  The  leukocytes  are  frequently  increased  to 
15,000.  In  the  anaemia  of  splenic  anaemia  there  is  a  marked  leuko- 
penia.  In  anaemias  from  malignant  tumors  the  color-index  is  usually 
of  the  chlorotic  type — the  haemoglobin  content  of  the  red  cells  being 
more  affected  than  the  number.  Normoblasts  are  usually  present,  and 
this  finding  may  differentiate  gastric  cancer  from  ulcer.  In  bone 
marrow  metastases  megaloblasts  may  be  expected.  Myelocytes  and 
so-called  tumor  cells  (large  cells  with  faintly-staining  vacuolated 
nuclei  and  but  little  cytoplasm)  may  also  be  found.  As  a  rule,  there 
is  a  moderate  leukocytosis  in  malignant  disease.  Eosinophiles  may  be 
largely  increased  in  sarcoma. 

THE  LEUKAEMIAS. 

It  is  in  the  leukaemias  that  we  have  the  greatest  increase  in  the  num- 
ber of  white  cells.  These  cases  show  more  or  less  anaemia,  but  we  may 
have  cases  of  myelogenous  leukaemia  showing  250,000  leukocytes  per 
cubic  millimeter  without  particular  change  in  the  red  cells.  The  more 
marked  the  red-cell  change  the  more  severe  the  condition. 

There  are  two  well-defined  types  of  leukaemia,  the  lymphatic  and  the 


MYELOGENOUS    LEUKEMIA.  167 

splenomyelogenous.  It  must  be  borne  in  mind,  however,  that  while 
a  greater  change  in  the  lymphatic  glands  may  produce  the  lymphatic 
type,  yet  even  in  such  cases  we  expect  to  find  alteration  in  bone  marrow 
and  spleen;  that  is,  there  is  a  general  involvement  of  the  haemopoietic 
system  in  all  leukaemias,  the  activity  being  most  marked  in  spleen  and 
bone  marrow  in  certain  cases  and  in  lymphatic  glands  in  others. 

Myelogenous  leukaemia  is  a  very  rare  disease,  about  five  times  as 
rare  as  pernicious  anaemia.  Lymphoid  leukaemia  is  still  more  rare. 

Splenomyelogenous  Leukaemia  (myeloid  leukaemia). — The 
differentiation  of  the  blood  picture  of  this  disease  from  leukocytosis 


FIG.  54. — Myelogenous  leukaemia,     m,  Myelocyte;  p,  polymorphonuclear; 
/,  mast  cell;  n,  normoblast.     (Cabot.) 

does  not  depend  on  the  number  of  leukocytes,  but  on  the  presence  and 
large  proportion  of  myelocytes.  We  expect  both  neutrophilic  and 
eosinophilic  myelocytes  in  myeloid  leukaemia — the  proportion  of  these 
varies,  but,  as  a  rule,  the  neutrophilic  one  is  the  common  one.  The 
blood  in  advanced  cases  is  milky  and  shows  a  most  marked  buffy  coat. 
The  marrow  is  largely  replaced  by  a  yellow  pyoid  material.  The 
spleen  may  weigh  ten  pounds.  The  leukocyte  count  is  on  the  average 
from  200,000  to  500,000.  Cases  are  recorded  of  more  than  one  million 
white  cells.  The  neutrophilic  myelocytes  make  up  about  thirty  to 
forty  per  cent,  of  these  and,  about  equal  in  number,  are  found  the 


1 68  NORMAL    AND    PATHOLOGICAL    BLOOD. 

polymorphonuclears,  while  the  percentage  of  the  lymphocytes  is 
decreased  (2  to  5%)  and  normal  eosinophiles,  eosinophilic  myelocytes, 
and  large  mononuclears  make  up  the  remaining  percentages.  We 
usually  have  great  numbers  of  normoblasts.  Megaloblasts  may  be 
rarely  found.  The  red  count  is  usually  about  2,500,000  and  the  color- 
index  low. 

Lymphatic  Leukaemia. — In  this  we  have  glandular  enlargements, 
but  not  such  large  masses  as  in  Hodgkin's  disease.  The  red  cells  are 
usually  reduced  about  one-half  and  the  color-index  is  a  little  below 
normal.  Normoblasts  are  rarely  found.  Myelocytes,  as  a  rule,  are 


FIG.  55. — Lymphatic  leukaemia,     p,  polymorphonuclear;  m,  megaloblast; 
e,  eosinopm'le.     Twenty-one  lymphocytes  in  this  field.     (Cabot.} 

absent,  but  may  amount  to  5%  of  the  leukocytes.  The  predominating 
leukocyte  (75  to  98%)  is  the  small  lymphocyte.  In  acute  lymphatic 
leukaemia  the  large  lymphocytes  predominate.  The  leukocyte  count  is 
never  so  great  as  in  myeloid  leukaemia,  rarely  exceeding  125,000. 

Hodgkin's  disease  is  usually  considered  as  a  disease  with  marked 
glandular  enlargements,  but  with  a  negative  blood  picture.  Undoubt- 
edly the  view  that  so-called  lymphosarcomata,  lymphatic  leukaemia  and 
Hodgkin's  disease  merge  into  one  another  and  that  they  represent  a 
malignant  cell  formation  in  the  haemopoietic  system  is  the  conservative 
one  to  take. 


NOTES  ON  BLOOD  WORK. 


NOTES  ON  BLOOD  WORK. 


NOTES  ON  BLOOD  WORK. 


NOTES  ON  BLOOD  WORK. 


PART  III. 
ANIMAL  PARASITOLOGY. 


CHAPTER  XV. 

GENERAL  CONSIDERATIONS  OF  CLASSIFICATION  AND 

METHODS. 

ANIMALS  that  are  in  all  respects  alike  we  term  a  Species.  Of  course 
the  male  and  female  of  a  species  may  be  very  unlike,  but  as  a  result  of 
mating  they  produce  young  having  characteristics  similar  to  the 
parents.  Now,  if,  as  in  the  case  of  the  mosquitoes  causing  yellow  fever, 
we  find  some  with  straight  silvery  lines  and  others  uniformly  showing 
crescentic  silvery  bands  about  thorax,  yet  resembling  each  other  closely 
in  the  respect  of  being  dark,  brilliantly-marked  mosquitoes,  we  should 
consider  them  as  being  separate  species  with  a  certain  relationship  to 
which  the  term  Genus  is  applied.  In  naming  a  species  we  always  first 
write  the  name  of  the  genus,  which  has  a  Greek  or  Latin  name,  com- 
mencing with  a  capital,  and  follow  with  the  specific  term,  which  latter 
commences  with  a  small  letter.  Thus  we  designate  the  dark  silver- 
marked  mosquitoes  as  belonging  to  the  genus  Stegomyia;  those 
showing  the  characteristics  of  curved  silver  bands  on  dorsal  surface  of 
thorax  we  designate  as  Stegomyia  calopus ;  the  species  with  the  straight 
silver  lines  we  call  Stegomyia  scutellaris. 

Again,  certain  genera  show  resemblances  which  enable  us  to  make 
broader  groupings  to  which  we  apply  the  name  Subfamily.  Thus  the 
genus  Stegomyia  and  the  genus  Culex  have  the  similar  characteristics  of 
palpi  in  the  female  being  shorter  than  the  straight  proboscis ;  we  there- 
fore classify  all  species  of  Stegomyia  and  all  species  of  Culex  under  the 
designation  Culicinae.  The  name  of  a  subfamily  ends  in  "inae."  Now, 
12  169 


170  CONSIDERATIONS    OF    CLASSIFICATION    AND    METHODS 

again,  certain  insects  are  different  from  others  in  having  scales  on  the 
wings.  We  find  that  not  only  do  the  Culicinae  have  such  character- 
istics, but  the  same  is  observed  with  the  Anophelinae  and  other  similar 
scale-wing  insects.  All  of  these  we  term  a  Family  and  we  speak  of  the 
Culicidae,  meaning  the  family  of  mosquitoes.  The  name  of  a  family 
ends  in  "idae."  Many  families  are  not  subdivided  into  subfamilies, 
but  are  directly  separated  into  genera.  Again,  a  genus  may  have  only 
a  single  species.  When  there  are  a  number  of  families  agreeing 
closely  in  some  striking  characteristic,  we  group  them  together  into  an 
Order;  thus,  the  family  of  mosquitoes  closely  resembling  many  other 
families  of  insects  in  possessing  a  pair  of  well-developed  wings  are 
grouped  in  the  order  Diptera;  all  of  which  resemble  certain  other 
animals  in  the  possession  of  a  distinct  head,  thorax  and  abdomen  with 
3  pairs  of  legs  projecting  from  the  thorax.  This  collection  of  animals 
we  call  a  Class;  thus,  we  speak  of  the  class  Insecta.  It  will  be  observed 
that  the  insects  have  no  internal  skeleton,  but  instead  a  chitinous 
cuticle,  the  exoskeleton.  Spiders,  ticks,  etc.,  resemble  them  in  this 
respect,  and  we  now  apply  to  all  such  animals  the  wider  designation, 
Branch  or  Phylum.  Inasmuch  as  the  animal  kingdom  is  divided  into 
the  branches  Protozoa,  Porifera,  Ccelenterata,  Echinodermata, 
Vermes,  Arthropoda,  Mollusca  and  Chordata,  we  see  that  the  branch  is 
the  largest  grouping  we  employ.  To  descend  in  the  scale  we  have 
belonging  to  the  branch,  the  classes;  to  the  class,  the  orders;  to  the 
order,  the  families;  to  the  family,  the  subfamilies;  to  the  subfamily, 
the  genera;  to  the  genus,  the  species.  Occasionally  a  species  is 
further  divided  into  subspecies. 

There  are  certain  terms  employed  in  animal  parasitology  which  it 
is  necessary  to  understand.  Among  these  we  shall  refer  to  the  follow- 
ing: 

i.  True  Parasitism.  By  this  is  understood  the  condition  where 
the  parasite  does  harm  to  the  host,  deriving  all  the  benefit  of  the 
association.  A  good  example  of  this  would  be  the  hook-worm  infecting 
man  or  animals. 

2.!*Mutualism.  In  such  an  association  there  is  mutual  benefit  to 
each  party  of  the  association.  An  instance  of  this  would  be  the 
presence  of  colon  bacilli  in  the  intestines.  The  bacillus  is  furnished 


ZOOLOGICAL  -NOMENCLATURE.  I  7 1 

a  suitable  habitat  and  in   return  protects    its  host   against   strictly 
pathogenic  bacteria. 

3.  Commensalism.     Here  there  is  benefit  to  the  parasite,  but  no 
injury  to  the  host.     An  example  of  this  kind  would  be  furnished  in  the 
case  of  the  Trichomonas  vaginalis  which  lives  in  the  vaginal  mucus, 
but,  so  far  as  known,  does  no  injury  to  the  host. 

4.  Nomenclature.  When  the  thousands  of  different  species,  genera, 
etc.,    of   animals   is   considered,  it   will  be   readily   perceived    that, 
unless  some  system  existed  for  their  designation,  indescribable  con- 
fusion would  prevail.     To  avoid  this,  the  International  Code,  based  on 
the  rules  of  Linnaeus   (1751),   requires  Latin  or  Latinized  names. 
There  are  certain  rules  governing  the  naming  of  animals.     Of  these, 
the  law  of  priority  provides  that  the  oldest  name,  under  the  code,  of 
any  genus  or  species  is  its  proper  zoological  name.      The  history  of  the 
naming  of  the  organism  of  syphilis  illustrates  this  well.     Schaudinn 
gave  this  organism  in  1905  the  name  of  Spirochaeta  pallida.     Ehren- 
burg,  in  1838,  had  used  the  name  Spirochaeta  for  animals  of  a  different 
character,  so  that  this  designation  of  the  genus  was  not  permissible 
under  the  code.     Villemin,  a  little  later,  proposed  the  name  of  Spiro- 
nema.     This  term,  however,  was  found  to  have  been  used  in  1864. 
Consequently  it  was  not  available.     Stiles  then  proposed  the  name 
Microspironema  but  as  Schaudinn  only  about  two  weeks  before  had 
offered  the  designation  Treponema,  the  name  Treponema  pallidum 
had  to  be  accepted  as  the  proper  zoological  name  for  the  organism  of 
syphilis. 

Another  point  is  that  names  are  not  definitions,  consequently  the 
fact  of  lack  of  appropriateness  of  any  name  is  no  objection  to  its  con- 
tinuation. This  will  appeal  to  anyone  as  a  wise  provision,  because  if  a 
different  name  were  substituted  each  time  a  designation  more  descrip- 
tive or  applicable  were  invented  it  would  be  utterly  destructive  to 
system.  When  it  is  considered  that  some  of  our  parasites  have  approxi- 
mately 50  different  designations,  for  the  most  part  given  by  medical 
observers,  it  will  be  appreciated  how  much  the  zoologist  has  aided  us 
in  trying  to  eliminate  all  but  the  single  proper  zoological  name. 

The  objections  so  frequently  heard  among  physicians  in  connection 
with  adopting  new  names  for  old  ones  are  not  well  founded.  Wher- 


172  CONSIDERATIONS    OF    CLASSIFICATION   AND    METHODS. 

ever  confusion  has  reigned,  the  establishment  of  order  always  results  in 
temporary  greater  confusion.  There  is  no  doubt  that  the  student 
taking  up  this  subject  a  few  years  hence  will  have  the  satisfaction, 
thanks  to  the  zoolo'gist,  of  only  having  to  burden  his  mind  with  one 
name^for  one  parasite. 

5.  Terminology.  This  applies  to  appropriate  designations  for 
different  organs,  symptoms,  etc.,  and  is  not  subject  to  any  rule  other 
than  that  of  good  usage. 


Class 


CHAPTER  XVI. 
THE  PROTOZOA. 

CLASSIFICATION  OF  PROTOZOA 

Order 


Gmnamceba- 


Genus 

Species 

• 

f  E.  coli 

Entamceba 

j   E.  histolyticax 
[  E.  buccalis 

Leydenia 

L.  gemmipara 

*• 

S.  recurrentis 

S.  vincenti 

Spirochaeta 

S.  duttoni 

S.  carteri 

S.  refringens 

Treponema 

Vv/V 

T.  pallidum 
T.  pertenue     *, 

Trypanosoma 

l^a*     ' 

Tnchomonas 

.  T.  gambiense~ 
f  T.  vaginalis 
\  T.  intestinalis 

Lamblia 

L.  intestinalis 

Babesia 

B.  bigemina 

Leishmania 

.      ^-fKcWg, 

f  L.  donovani 
\  L.  tropica 

Balantidium 

B.  coli 

E.  stiedae 

Isospora 

I.  bigemina 

(P.  vivax 

Plasmodium 

P.  malaria? 

-J, 

P.  falciparum 

X 


Rhkopoda 

(Sarcodina) 

These  throw  out  proto- 
plasmic projections  called 
pseudopodia. 


Flagella'ta 

(Mastigophora) 
These  move  by  means 
of  undulating  membranes 
or  flagella. 


Infusoria  Heterotricha 

(Ciliata) 

These  have  contractile 
vacuoles  and  numerous 
fine  cilia  which  are  shorter 
than  flagella  and  have  a 
sweeping  stroke. 

Spopozoa  Coccidiaria 

These  have  no  motile 
organs.  They  live  para- 
sitically  in  the  cells  or 
tissues  of  other  animals.  Haemosporidia 
Reproduction  by  spores. 

THE  PROTOZOA. 

RmzopoDA  (SARCODINA). 

In  this  class  of  protozoa  the  pseudopodia  serve  the  double  purpose 
of  nutrition  and  locomotion.     These  protoplasmic  extensions  may  be 

173 


174 


THE    PROTOZOA 


quite  broad  or  very  narrow.  As  a  rule,  the  thicker  the  pseudopod  the 
more  rapid  the  movement.  Some  rhizopods  have  hard  shell-like 
coverings  which  are  secreted  in  or  on  the  ectosarc.  These  skeletons 
have  openings  through  which  the  pseudopods  project.  The  pseudopo- 
dia  may  be  made  up  only  of  ectoplasm  or  both  ectoplasm  and  endoplasm 
may  take  part.  Amoeboid  movement  always  starts  in  the  ectoplasm.  In 


FIG.  56. — Various  protozoa.  i,  Entamoeba  coli;  2,  Entamoeba  histolytica; 
3,  Leydenia  gemmipara;  4,  Trichomonas  vaginalis;  5,  Trichomonas  intestinalis; 
6,  Lamblia  intestinalis;  7,  flagellated  Leishmania  donovani;  8,  Leishmania  donovani 
in  phagocyte;  9,  Eimeria  stiedae;  10,  Isospora  bigemina;  n,  Trypanosoma  gam- 
biense;  12,  Balantidium  coli. 

addition  to  the  nucleus,  which  the  so-called  chromatin  staining  methods 
bring  out  as  reddish  areas,  we  frequently  observe  smaller  aggregations 
of  chromatin  staining  material  in  the  cytoplasm.  This  extranuclear 
chromatin  is  supposed  to  play  a  part  in  the  more  intricate  divisions 
which  such  protozoa  undergo.  Food  vacuoles  and  contractile  vacuoles 
are  present  in  many  rhizopods. 


INTESTINAL  AMCEB^.  175 

Entamoeba  coli. — This  is  considered  by  Schaudinn  to  be  a  harm- 
less inhabitant  of  the  intestines  and  its  presence  in  the  faeces  is  not 
considered  of  importance.  Musgrave  and  Clegg  do  not  recognize  a 
distinction  between  a  nonpathogenic  and  a  pathogenic  amoeba,  but 
consider  that  the  presence  of  amoebae,  in  the  absence  of  symptoms,  is 
to  be  explained  by  the  nonestablishment  of  a  satisfactory  symbiosis 
with  some  bacterium  or  other  parasite.  They  state,  that  as  a  result  of 
extended  observation,  persons  harboring  amoebae  will  sooner  or  later 
develop  dysentery.  As  regards  the  morphological  points  of  distinc- 
tion, they  state  that  even  in  pure  cultures,  descended  from  a  single 
amoeba,  the  same  variations  in  size  motility,  etc.,  may  be  observed. 
They  also  consider  that  amoebae  having  all  the  characteristics  of  the 
harmless  commensal  may  cause  marked  pathological  change.  Craig 
claims  that  the  E.  coli  cannot  be  cultivated  and,  that  several  years 
since  noting  E.  coli  in  stools  of  healthy  persons,  these  persons  have 
remained  free  of  any  dysenteric  symptoms. 

The  only  safe  way  in  recognizing  amoebae  in  stools  is  to  note 
amoeboid  movement.  The  encysted  amoebae  can  scarcely  be  differ- 
entiated from  many  vegetable  cells  and  especially  from  large  phago- 
cvtic  cells,  of  probable  endothelial  origin.  By  the  use  of  neutral  red  in 
very  dilute  solution  the  granular  endoplasm  will  be  observed  to  take 
up  the  brick-red  stain. 

E.  coli  varies  greatly  in  size  (8  to  40/1).  There  is  no  well-marked 
distinction  between  a  granular  interior  and  a  more  compact,  hyaline 
exterior.  The  nucleus  is  centrally  situated,  is  distinct  and  on  staining 
with  Wright's  stain  shows  the  chromatin  coloration.  It  is  sluggishly 
motile  and  is  of  a  grayish-white  color.  When  stained  it  does  not 
show  a  distinction  between  endoplasm  and  ectosarc.  The  infecting 
stage  is  an  encysted  form  with  8  amcebulae. 

Entamceba  histolytica. — This  is  considered  the  pathogenic 
amceba.  Schaudinn  considers  that  it  is  by  the  possession  of  its 
tough,  tenacious,  glassy  and  highly  refractile  ectoplasm  that  it  is  able 
to  bore  its  way  into  the  submucosa  of  the  large  intestine  and  bring 
about  those  gelatinous  like  necroses,  which,  by  undermining,  even- 
tually result  in  dysenteric  ulcerations. 

It  is  also  the  species  found  in  tropical  liver  abscess.     As  described 


176  THE  PROTOZOA 

by  Schaudinn,  it  has  a  marked  differentiation  between  the  glassy 
ectoplasm  and  the  granular  endoplasm.  The  nucleus  is  indistinct, 
eccentric  and  stains  feebly.  The  movement  is  more  active  and  the 
color  more  greenish-yellow  than  E.  coli.  Craig  notes  the  character- 
istic staining  of  the  E.  histolytica,  this  being  a  dark  blue  ectoplasm 
encircling  a  lighter  blue  endoplasm.  In  dividing,  there  is  a  process  of 
budding.  These  little  spore-like  bodies  form  at  the  periphery  of  the 
encysted  amoeba  and  form  the  infecting  stage.  Cold  or  other  inimical 
influences  tend  to  make  amoebae  encyst,  hence  faeces  should  be  ex- 
amined as  soon  as  possible  after  the  stool  is  passed.  A  particle  of 
mucus  pressed  down  with  a  cover-glass  makes  a  satisfactory  prepara- 
tion. If  necessary  to  dilute,  use  blood-warm  salt  solution — not  plain 
water. 

Entamoeba  buccalis. — Obtained  from  the  mouths  of  persons 
with  dental  caries.  It  does  not  appear  to  have  pathogenic  character- 
istics. 

Castellani  has  reported  an  intestinal  amoeba  with  an  undulatory 
membrane.  He  has  given  it  the  name  of  E.  undulans. 

Leydenia  gemmipara. — It  is  a  question  whether  these  bodies  were 
animal  parasites  or  simply  body  cells  showing  amoeboid  movement. 
They  were  found  in  the  ascitic  fluid  of  2  cases  of  carcinomatosis. 
They  varied  in  size  from  3  to  36  //. 

FLAGELLATA  (MASTIGOPHORA). 

In  this  class  of  protozoa  the  adults  have  flagella  for  the  purposes 
of  locomotion  and  the  obtaining  of  food. 

Some  flagellates  more  or  less  resemble  rhizopods  in  being  amoeboid 
and  in  having  an  ectoplasm  and  an  endoplasm.  The  body  is  fre- 
quently covered  by  a  cuticle.  Some  flagellates  have  a  definite  mouth 
part,  the  cytostome,  which  leads  to  a  blind  oesophagus ;  others  absorb 
food  by  absorption  through  the  body  wall.  In  addition  to  flagella, 
some  flagellates  possess  an  undulating  membrane.  All  flagellates 
possess  a  nucleus  and  some  have  contractile  vacuoles.  The  flagellum 
may  arise  directly  from  the  nucleus  or  from  a  small  kinetic  nucleus, 
the  blepharoplast. 

The   most  important  flagellates  of  man  are  the  haemoflagellates. 


RELAPSING   FEVER.  177 

Among  these  we  may  include  the  blood  spirochaetes  and  the  organism 
of  syphilis,  which  have  many  resemblances  to  the  spiral  form  of  bac- 
teria, together  with  the  three  genera  in  which  protozoal  characteristics 
are  marked,  namely,  Leishmania,  Trypanosoma  and  Trypanoplasma. 
In  addition  we  have  flagellates  in  the  intestinal  canal  and  in  the  vaginal 
secretion.  Some  authors  place  the  genus  Piroplasma  with  the  flagel- 
lates and  there  has  been  controversy  concerning  the  nature  of  certain 
projections  from  these  bodies.  It  would  seem  preferably  however,  to 
consider  them  under  the  sporozoa. 


FIG.  57. — Spirilla  of  relapsing  fever  from  blood  of  a  man. 
(Kolle  and  Wassermann.) 


Spirochaeta. 

The  generic  term  Spirochaeta  is  applied  to  flagellates  having  a 
spiral  shape,  an  undulating  membrane  and  no  flagella.  This  genus  is 
one  about  which  there  are  two  views:  one,  that  the  members  belong  to 
the  bacteria;  the  other,  that  they  are  protozoa.  The  absence  of 
demonstrable  nucleus  and  blepharoplast  make  them  apparently 
vegetable  in  nature  while  the  variations  in  thickness,  the  fact  of  trans- 
mission by  an  arthropod  and  indications  of  a  longitudinal,  rather  than 
a  transverse  division,  would  indicate  protozoal  affinities. 

S.  recurrentis.— This  is  the  organism  of  relapsing  fever.     It  was 


178  THE    PROTOZOA. 

formerly  considered  a  bacterium  and  was  termed  the  Spirochaeta 
obermeieri.  It  is  present  in  the  blood  of  persons  suffering  from  the 
disease  during  the  pyrexia.  During  the  apyrexia  they  are  not  found  in 
the  peripheral  circulation.  At  this  time  they  are  present  in  great 
numbers  in  the  spleen  where  they  are  actively  phagocytized.  The 
disease  is  supposed  to  be  transmitted  by  bed-bugs  or  lice.  Monkeys 
are  susceptible  and,  after  passage  through  monkeys,  rats  can  be 
inoculated. 

S.  duttoni. — This  is  the  cause  of  South  African  tick  fever  or  Tete 
fever.  The  disease  is  similar  to  relapsing  fever,  but  there  are  generally 
four  or  five  febrile  paroxysms  with  apyrexial  intervals.  The  disease  is 
readily  transmitted  to  ordinary  laboratory  animals,  especially  the  rat. 
Rats  which  have  recovered  from  S.  recurrentis  can  be  infected  by 
S.  duttoni  and  vice  versa.  The  disease  is  transmitted  by  the  bite 
either  of  the  adult  or  larval  Ornithodoros  moubata.  Koch  found 
spirochaetes  in  the  eggs  of  the  ovaries  of  ticks  which  had  fed  on  persons 
with  the  disease.  It  is  thus  an  instance  of  hereditary  transmission. 

Other  spirochaetes  that  have  been  considered  as  pathogenic  for  the 
type  of  relapsing  fever  in  India  and  that  of  America  are  the  S.  carteri 
and  the  S.  novyi. 

S.  vincenti. — This  is  a  very  delicate  spiral-shaped  organism 
which  has  been  found  in  conjunction  with  a  fusiform  bacillus  in  a 
throat  inflammation,  usually  termed  Vincent's  angina. 

S.  refringens. — This  Spirochaeta  is  frequently  associated  with  the 
Treponema  pallidum  and  is  common  in  genital  ulcerations.  It  is 
thicker,  has  less  regular  and  more  flattened  curves  and  stains  more 
readily. 

Treponema. 

The  genus  Treponema  has  no  undulating  membrane  and  has  a 
flagellum  at  each  end. 

Treponema  pallidum. — This  is  the  cause  of  syphilis.  It  is  char- 
acterized by  its  very  geometric  regularity  in  the  spirals,  which  are 
deeply  cut,  and  in  focussing  up  and  down  continue  in  focus  (like  a 
corkscrew).  They  require  from  one  to  two  hours  to  stain  distinctly 
with  Giemsa's  stain  and  the  attenuated  ends  or  flagella  should  always 


YAWS. 


179 


be  noted  before  reporting  their  presence.     To  stain  them  in  section 
Levaditi's  method  is  the  best. 

T.  pertenue. — An  organism  of  similar  morphology  was  first 
reported  by  Castellani  as  present  in  yaws.  It  is  found  in  smears  and 
sections  as  with  T.  pallidum. 


FIG.  58. — The  spirochaeta  refringens  is  the  larger  and  more  darkly  stained 
organism,  while  the  lightly  stained  and  more  delicate  parasites  is  the  Spirochaeta 
pallida.  (Treponema  pallidum).  From  a  chancre  stained  with  Wright's  blood 
stain.  (Hirsch — by  Rosenberger.) 


Trypanosoma. 

The  genus  Trypanosoma  has  a  more  or  less  spindle-shaped  body, 
along  one  border  of  which  runs  an  undulating  membrane.  There  is 
one  flagellum  bordering  the  membrane  and  projecting  like  a  whip 


l8o  THE    PROTOZOA. 

posteriorly.  There  is  a  nucleus  (macronucleus)  and  a  blepharoplast 
(micronucleus — centrosome),  the  latter  being  located  anteriorly  as  a 
chromatin  staining  dot  or  rod.  From  this  blepharoplast  the  flagellum 
proceeds  posteriorily  bordering  the  undulating  membrane  and  pro- 
jecting freely  beyond  the  posterior  end.  The  nucleus  is  larger, 
nearer  the  posterior  end,  and  does  not  stain  so  intensely  as  the 
blepharoplast. 

T.  gambiense. — This  is  the  trypanosome  causing  human  trypano- 
somiasis,  the  latter  stage  of  which  is  known  as  sleeping  sickness.  It  is 
from  17  to  28/1  long,  and  from  1.5  to  2/z  wide. 

It  is  very  difficult  to  distinguish  the  human  trypanosome  from  some 
of  the  other  pathogenic  ones  by  staining  methods.  The  immunity 
test  is  the  most  reliable.  An  animal  recovered  from  an  infection  by  a 
certain  trypanosome  does  not  possess  immunity  for  other  pathogenic 
ones.  Novy  and  McNeal  cultivated  T.  lewisi  in  water  of  condensation 
on  blood  agar,  but  up  to  the  present  T.  gambiense  has  not  been  culti- 
vated. It  is  present  in  the  blood,  usually  in  exceedingly  small  numbers, 
and  in  the  lymphatic  glands  of  patients.  It  is  by  puncture  of  the 
glands  that  we  have  the  best  means  of  finding  the  parasites.  It  is  also 
found  in  the  cerebrospinal  fluid  in  sleeping  sickness.  The  parasite 
stains  readily  with  Wright's  stain.  The  transmitting  agent  is  the 
Glossina  palpalis.  It  is  not  known  whether  this  occurs  by  direct  or 
indirect  transmission.  At  any  rate,  there  must  be  some  peculiarity 
about  the  tsetse  fly,  either  in  the  reaction  of  its  salivary  secretion  or 
otherwise,  to  make  it  the  only  well-recognized  agent  in  the  spread  of 
the  disease.  No  tsetse  fly,  no  trypanosomiasis.  Koch  observed 
certain  developmental  forms  in  the  bulb  of  the  proboscis,  but  whether 
they  represented  a  developmental  cycle  or  not  is  not  settled. 

Other  authorities  think  it  possible  that  trypanosomes  may  encyst  in 
the  digestive  tract  and  so  the  flies  transmit  the  disease  along  with  their 
faeces.  This  does  not  seem  to  be  possible  in  connection  with  human 
infections.  Koch  found  several  cases  where  infection  had  taken  place 
by  coitus.  This  is  the  method  of  infection  in  T.  equiperdum,  a 
trypanosome  disease  of  horses. 

Of  the  more  important  trypanosome  diseases  of  animals  may  be 
mentioned : 


KALA  AZAR.  l8l 

1.  Nagana.     Pathogenic  for  domestic  animals.     T.  brucei. 

2.  Surra.     Pathogenic  for  horses  in  India  and  Philippines. 
T.  evansi. 

3.  Dourine.  Transmitted  by  coitus  in  horses.     T.  equiper- 
dum. 

4.  Mai  de  caderas.     Affects  horses  in    South  America.     T. 
equinum. 

A  harmless  infection,  especially  in  sewer  rats,  is  due  to  T.  lewisi. 
There  are  many  trypanosomes  in  birds,  fish,  frogs,  etc. 

Trypanoplasma. 

The  genus  Trypanoplasma  has  a  rather  large  blepharoplast, 
from  which  arise  two  flagella.  One  extends  forward  as  a  free  anterior 
flagellum,  while  the  other  projects  posteriorly,  running  along  the 
border  of  the  undulating  membrane.  This  Genus  is  not  known  for 
man. 

Leishmania. 

The  genus  Leishmania  includes  two  species:  one,  the  L.  donovani, 
the  parasite  of  kala  azar,  and  the  other  the  L.  tropica,  the  parasite 
of  oriental  sore.  These  are  undoubtedly  different  species,  inasmuch 
as  in  sections  of  India,  where  tropical  ulcer  was  common,  there  was 
no  kala  azar,  and  in  Assam  where  kala  azar  prevailed  there  were  no 
Leishman-Donoyan  bodies  to  be  found  in  smears  from  the  tropical 
ulceration  there  present,  except  rarely  in  cases  of  general  infection. 
L.  tropica  has  not  been  cultivated. 

These  parasites  are  typically  intracellular,  being  within  either 
polymorphonuclears,  which  contain  only  one  or  two  of  the  bodies,  or  in 
large  mononuclears,  in  which  there  may  be  as  many  as  six.  They 
may  be  packed,  however,  in  phagocytic  endothelial  cells.  The  parasites 
occur  in  the  peripheral  circulation  in  about  80%  of  the  cases.  They 
abound  in  the  liver  and  spleen.  The  parasite  is  oval  and  about  2x3^. 
There  are  two  distinct  chromatin  staining  masses.  The  larger  nu- 
cleus is  more  or  less  spherical  and  stains  faintly,  while  the  smaller 
chromatin  mass  is  generally  rod-shaped  and  stains  intensely.  It  has 
been  recently  recommended  that  instead  of  liver  or  splenic  puncture 


1 82  THE    PROTOZOA. 

for  the  demonstration  of  these  bodies,  a  blister  be  raised  and  a  smear 
from  that  containing  many  polymorphonuclears  might  show  these 
bodies.  The  affection  is  characterized  by  a  leucopaenia  so  that  it  is 
very  difficult  to  demonstrate  the  parasites  in  ordinary  blood  smears. 
By  cultivating  the  parasites  obtained  from  splenic  puncture  in 
acidified  sodium  citrate  solution  at  room  temperature,  Rogers  suc- 
ceeded in  obtaining  flagellated  forms  similar  to  Herpetomonas. '  An 
anterior  flagellum  proceeds  directly  from  the  blepharoplast.  The 
bed-bug  is  supposed  to  be  the  intermediary  host. 

Trichomonas. 

Trichomonas  vaginalis. — This  parasite  has  a  fusiform  body  and 
is  about  18  x  10/1.  It  has  three  flagella  arising  from  the  anterior  end 
and  an  undulating  membrane.  It  lives  in  vaginal  mucus  which  has  an 
acid  reaction.  A  change  of  reaction,  as  at  menstrutation,  causes  them 
to  disappear.  Forms  similar  to  the  T.  vaginalis  have  been  found  in 
the  intestine  and  in  sputum  from  putrid  bronchitis. 

These  flagellates  are  generally  considered  harmless,,  although 
doubt  as  to  this  is  expressed  by  some  authors. 

Lamblia. 

Lamblia  intestinalis. — These  parasites  are  about  10  x  15^  and 
have  a  pear  shaped  body  with  a  depression  at  the  blunt  anterior  end. 
This  depression  enables  the  flagellate  to  attach  itself  to  the  summit  of 
an  epithelial  cell.  Around  the  depression  are  three  pairs  of  flagella 
which  are  constantly  in  motion.  Another  pair  of  flagella  project  from 
either  side  of  the  blunt  little  tail-like  projection.  When  stained,  the 
parasites  have  a  pyriform  shape  with  two  chromatin  staining  areas  on 
either  side  of  the  anterior  end.  When  encysted,  they  assume  a  circular 
shape.  This  parasite  is  generally  considered  as  of  little  importance, 
but  inasmuch  as,  when  in  great  numbers  in  the  csecum  and  appendix, 
they  may  give  rise  to  symptoms  resembling  appendicitis  and  as  they 
are  responsible  for  a  chronic  and  intractable  diarrhoea  associated  with 
mental  and  physical  depression,  this  is  undoubtedly  an  affection  only 
minor  in  importance  to  amoebic  infection.  It  is  a  very  common 
infection  in  the  tropics. 


SPOROZOA.  183 

INFUSORIA  (CILIATA.) 

The  bodies  of  Infusoria  are  oval  and  may  be  free  or  attached  to  a 
stalk-like  contractile  pedicle,  as  with  Vorticella,  or  they  may  be  sessile. 
The  cilia,  which  are  characteristic,  may  be  markedly  developed  around 
the  cystostome  (mouth)  and  serve  the  purpose  of  directing  food  into  the 
interior,  while  others  act  as  locomotor  organs.  The  body  is  enveloped  by 
a  cuticle  which  may  only  have  one  opening  or  slit,  to  serve  as  mouth ; 
or  it  may  have  a  second  one,  a  cytopyge  or  anus.  Usually  the  fecal 
matter  is  ejected  through  a  pore  which  may  be  visible  only  when  in  use. 
They  usually  have  a  large  nucleus  and  a  small  one.  Infusoria  tend  to 
encyst  when  conditions  are  unfavorable. 

Balantidium  coli. — This  is  the  only  ciliate  of  importance  in  man. 
It  is  a  common  parasite  of  hogs.  It  is  from  60  to  loofj.  long  by  50  to 
70 ,«  broad,  and  has  a  peristome  at  its  anterior  end  which  becomes 
narrow  as  it  passes  backward.  It  has  an  anus.  The  ectosarc  and  the 
endorsarc  are  distinctly  marked.  These  parasites  cause  an  affection 
similar  to  dysentery  and  may  bring  about  a  fatal  termination.  It  is 
almost  impossible  to  escape  noticing  the  actively  moving  bodies  if  a 
fecal  examination  is  made.  When  encysted  they  are  round. 

Another  ciliate,  the  Nyctotherus  faba,  has  a  kidney -shaped  body 
and  is  about  25  by  15;*.  It  has  a  large  contractile  vacuole  at  the 
posterior  end.  It  has  a  large  nucleus  in  the  center  with  a  small  fusi- 
form micronucleus  lying  close  to  it.  It  has  only  been  reported  once  for 
man. 

SPOROZOA. 

This  class  of  protozoa  gets  its  name  from  the  method  of  reproduc- 
tion— sporulation.  These  parasites  rarely  show  binary  fission. 
While  the  sporozoa  are  found  within  cells,  in  the  tissues  and  in 
internal  cavities,  as  intestine  and  bile  ducts,  yet  it  is  as  inhabitants  of 
the  blood  that  they  have  their  greatest  importance  for  man — these  are 
known  as  Haemosporidia.  A  sporozoon  may  be  either  naked  or 
amoeboid  or  be  covered  with  a  distinct  cuticle. 

Coccidiaria. 

The  parasites  of  the  order  Coccidiaria  are  almost  exclusively  found 
in  the  intestines  and  in  the  organs  connected  with  it.  In  the  vegetative 


DESCRIPTION  OF  PLATE  I. 

(Kolle  and  Wassermann.) 

Malarial  Parasites. 

1.  Two  tertian  parasites  about  thirty-six  hours  old,  attacked  blood-cor- 
puscles magnified. 

2.  Tertian  parasite  about  thirty-six  hours  old;  stained  by  Romanowsky's 
method.     The  black  granule  in  the  parasite  is  not  pigment  but  chromatin. 
Next  to  it  and  to  the  left  is  a  large  lymphocyte,  and  under  it  the  black  spot  is  a 
blood  plate. 

3.  Tertian  parasite,  division  form  nearby  is  a  polynuclear  leukocyte. 

4.  Quartan  parasite,  ribbon  form. 

5.  Quartan  parasite,  undergoing  division. 

6.  Tropical  fever  parasite.     (^Estivo-autumnal.)     In  one  blood -corpuscle 
may  be  seen  a  smaller,  medium,  and  large  tropical  fever-ring  parasite. 

7.  Tropical  fever  parasite.     Gametes  half  moon  spherical  form.     Smear 
from  bone  marrow. 

8.  Tropical  fever  parasite  which  is  preparing  for  division  heaped  up  in  the 
blood  capillaries  of  the  brain. 

Asexual  Forms. 

9.  Smaller  tertian  ring  about  twelve  hours  old. 

10.  Tertian  parasite  about  thirty-six  hours  old,  so-called  ameboid  form. 

11.  Tertian  parasite  still  showing  ring  fever,  forty-two  hours  old. 

12.  Tertian  parasite,  two  hours  before  febrile  attack.     The  pigment  is 
beginning  to  arrange  itself  in  streaks  or  lines. 

13.  Tertian  parasite  further  advanced  in  division.     Pigment  collected  in 
large  quantities. 

14.  Further  advanced  in  the  division.     (Tertian  parasite.) 


PLATE  I. 


MALARIA.  185 

stage  it  lives  within  an  epithelial  cell,  which  it  destroys.  Afterward 
it  falls  into  the  lumen  lined  by  this  epithelial  cell  and  sporulates,  either 
by  the  method  of  schizogony  or  sporogony. 

Eimeria  stiedae. — This  sporozoon  is  usually  known  as  the 
Coccidium  cuniculi  or  C.  oviforme.  It  is  most  frequently  found  in  the 
epithelium  of  the  bile  ducts.  It  has  very  rarely  been  reported  for  man. 
The  parasite  is  about  40  x  2o//,  and  is  oval  in  shape  with  a  double 
outlined  integument.  The  sporozoites,  which  form  inside,  are  falci- 
form in  shape.  These  escape  and  enter  fresh  epithelial  cells,  and  thus 
the  process  of  schizogony  goes  on.  The  parasites  of  the  liver  are 
larger  than  those  found  in  the  intestines,  these  latter  being  only  about 
30  x  i5//.  In  the  faeces  the  form  most  often  found  is  the  oocyst,  about 
40  x  25//. 

Isospora  bigemina. — This  parasite,  formerly  called  the  Coccidium 
bigeminum,  lives  in  the  intestinal  villi  of  dogs  and  cats.  It  is  about 
12  x  8fjL,  and  shows  a  highly  refractile  envelope  containing  two 
biscuit-shaped  spores.  It  has  been  reported  for  man. 

Haemosporidia. 

Of  the  sporozoa  found  in  the  blood  (Haemosporidia),  the  malarial 
parasites  are  the  only  ones  connected  with  disease  in  man. 

In  addition  to  man,  infections  with  parasites  of  a  similar  nature  are 
found  in  monkeys  (Plasmodium  kochi;  the  sexual  forms  alone  seem 
to  be  present),  in  birds  (PL  praecox;  this  infection  is  usually  desig- 
nated Proteosoma.  An  infection  of  crows  and  pigeons  of  like  nature  is 
Halteridium).  Numerous  haemosporidia  have  been  reported  for  bats, 
various  other  mammals,  tortoises,  lizards,  etc. 

The  life  history  of  the  malarial  parasite  is  one  of  the  most  interesting 
chapters  in  medicine.  Laveran  discovered  the  parasite  in  1880.  In 
1885,  Golgi  noted  that  sporulation  occurred  simultaneously  at  time  of 
malarial  paroxysm.  Koch,  Golgi  and  Celli  demonstrated  existence  of 
different  species  for  different  types  of  fever.  King  and  Laveran  (1884) 
considered  possibility  of  mosquito  transmission.  Manson  (1894) 
formulated  hypothesis  that  gametes  were  destined  to  undergo  develop- 
ment in  the  mosquito  from  observing  that  flagellated  bodies  only  ap- 
peared some  time  after  the  blood  was  withdrawn. 


DESCRIPTION  OF  PLATE  II. 

(Kolle  and  Wassermann.) 

Malarial  Parasites. 

15.  Complete  division  of  the  parasite.     Typical  mulberry  form. 

16.  To  the  left  is  the  completed  division  form,  an  almost  developed  gamete, 
which  is  to  be  recognized  by  its  dispersed  pigment. 

17.  A  tertian  ring  parasite,  small  size  broken  up. 

18.  Three-fold  infection  with  tertian  parasite.     The  oval  black  granules 
are  the  chromatin  granules. 

19.  To  the  left,  tertian  parasite  with  large,  sharply  demarked,  and  deeply 
colored  chromatin  granules.     To  the  right,  tertian  parasite.     Both  thirty-six 
hours  old.     Both  probably  gametes. 

20.  Tertian  parasite  thirty-six  hours  old,  ring  form. 

21.  Tertian  parasite  with  beginning  chromatin  division,  with  eight  chroma- 
tin  segments. 

22.  Tertian  parasite  chromatin  division  farther  advanced  with  twelve 
chromatin  granules,  in  part  triangular  in  form. 

23.  Completed  division  figure  of  a  tertian  parasite.     Twenty-two  chroma- 
tin  granules. 

24.  The  young  tertian  parasites  separating  themselves  from  each  other. 
The  pigment  remains  behind  in  the  middle. 

25.  Quartan  ring  parasite,  which  is  hard  to  differentiate  from  large  tropical 
ring  or  small  tertian  ring. 

26.  Quartan  ring  lengthening  itself. 

27.  Small  quartan  ribbon  form. 

28.  The  quartan  ribbon  increases  in  width.     The  dark  places  consist  al- 
most entirely  of  pigment. 


PLATE  II. 


MALARIA.  187 

Ross  (1895)  demonstrated  that  flagellation  takes  place  in  the 
stomach  of  the  mosquito.  McCallum  (1897)  saw  fertilization  of 
macrogametes  by  microgametes  of  Halteridium.  Opie  recognized 
differences  in  sexual  charactersistics. 

Ross  (1898)  demonstrated  life  cycle  of  bird  malaria  (Proteosoma), 
showing  formation  of  zygotes  and  presence  of  sporozoites  in  salivary 
glands.  Grassi  and  Bignami  proved  the  cycle  for  Anophelinae  for 
human  malaria.  In  1900  (Sambon  and  Low),  infected  mosquitoes 
from  Italy  were  sent  to  London,  where,  by  biting,  they  infected  two 
persons. 

Life  History. — When  man  is  at  first  infected  by  sporozoites  we  have 
starting  up  a  non-sexual  cycle  which  is  completed  in  from  forty-eight 
to  seventy-two  hours,  according  to  the  species  of  parasite.  The  falciform 
sporozoite  bores  into  a  red  cell,  assumes  a  round  shape  and  continues  to 
enlarge  (schizont).  Approaching  maturity,  it  shows  division  into  a 
varying  number  of  spore-like  bodies.  At  this  stage  the  parasite  is 
termed  a  merocyte.  When  the  merocyte  ruptures,  these  spore-like 
bodies  or  merozoites  enter  a  fresh  red  cell  and  develop  as  before. 

At  the  time  that  the  merocyte  ruptures  it  is  supposed  that  a  toxin  is 
given  off  which  causes  the  malarial  paroxysm.  The  cycle  goes  on  by 
geometric  progression  from  the  first  introduction  of  the  sporozoite,  but 
it  is  usually  about  two  weeks  before  a  sufficient  number  of  merocytes 
rupture  simultaneously  to  produce  sufficient  toxin  for  symptoms 
(period  of  incubation).  This  cycle  is  termed  schizogony. 

After  a  varying  time,  whether  by  reason  of  necessity  for  renewal  of 
vigor  of  the  parasite  by  a  respite  from  sporulation,  or  whether  from 
a  stand-point  of  survival  of  the  species,  sexual  forms  (gametes)  develop. 
Some  think  that  sporozoites  of  sexual  and  nonsexual  characteristics  are 
injected  at  the  same  time.  It  is  usually  considered,  however,  that  sexual 
forms  develop  from  preexisting  nonsexual  parasites. 

These  gametes  show  two  types:  the  one  which  contains  more  pig- 
ment, has  less  chromatin  and  stains  more  deeply  blue  is  the  female — a 
macrogamete;  the  other  with  more  chromatin,  less  pigment  and  stain- 
ing grayish-green  rather  than  blue  is  the  male — a  microgametocyte. 
When  the  gametes  are  taken  into  the  stomach  of  the  Anophelinae,  the 
male  cell  throws  off  spermatozoa -like  projections,  which  have  an  active 


DESCRIPTION  OF  PLATE  III. 

(Kolle  and  Wassermann). 

Malarial  Parasite. 

29>3°>3I-  The  quartan  ribbon  increases  in  width.  The  dark  places  con- 
sist almost  entirely  of  pigment. 

32.  Beginning  division  of  the  quartan  parasite  and  the  black  spot  in  the 
middle  is  the  collected  pigment. 

33-  Quartan  ring. 

34.  Double  infection  with  quartan  parasites. 

35.  Wide  quartan  band.     The  fine  black  stippling  in  the  upper  half  of  the 
parasite  is  pigment. 

36.  Beginning  division  of  the  quartan  parasite.     The  chromatin  (black 
fleck)  is  split  into  four  parts. 

37.  Division  advanced,  quartan  parasites. 

38.  Typical  division  figure  of  the  quartan  parasite. 

39.  Finished  division  of  the  quartan  parasite.     Ten  young  parasites,  pig- 
ment in  the  middle. 

40.  Young  parasites  separated  from  one  another. 

41.  Small  and  medium  tropical  ring,  the  latter  in  a  transition  stage  to  a 
large  tropical  ring. 

42.  Small,  medium  and  large  tropical  ring,  together  in  one  corpuscle. 


PLATE  III, 


MALARIA. 


189 


iashing  movement  and  break  off  from  the  now  useless  cell  carrier  and 
are  thereafter  termed  microgametes.  These  fertilize  the  macrogametes 
and  this  body  now  becomes  a  zygote. 

By  a  boring-like  movement  the  zygote  goes  through  the  walls  of  the 
mosquito's  stomach,  stopping  just  under  the  outer  epithelial  layer  of 
the  stomach  or  mid-gut.  It  continues  to  enlarge  until  about  the  end  of 


.7-0% . 


FIG.  59. — Sexual  and  non- sexual  cycle  of  malaria,  i,  Schizonts;  2,  merocyte; 
3,  merozoites;  4,  macrogamete;  5,  microgametocyte;  6  and  7,  gametes  in  stomach 
of  mosquito;  8,  microgametocyte  throwing  off  microgametes;  9,  microgamete  fer- 
tilizing macrogamete;  10,  vermiculus  or  zygote;  n  and  12,  Zygotes;  13,  Zygote 
distended  with  sporozoites;  14,  sporozoites. 

one  week  it  has  grown  to  be  about  60 /i  in  diameter  and  has  become 
packed  with  hundreds  of  delicate  falciform  bodies. 

Zygotes  of  benign  tertian  show  little  rod-like  particles  of  yellowish 
pigment — those  of  malignant  tertian  black  clumps,  which,  however,  are 
not  so  coarse  as  those  of  quartan. 

The  mature  zygote  now  ruptures  and  the  sporozoites  are  thrown  off 
into  the  body  cavity.  They  make  their  way  to  the  salivary  glands  and 


DESCRIPTION  OF  PLATE  IV. 

(Kolle  and  Wassermann.) 

Malarial  Parasite. 

43.  To  the  left  a  young  (spore)  tropical  parasite.     To  the  right  a  medium 
and  large  tropical  parasite. 

44.  An  almost  fully  developed  tropical  parasite.     The  black  granules  are 
pigment  heaps. 

45.  Young  parasites  separated  from  one  another.     Broken  up  division   . 
forms  twenty-one  new  parasites. 

46.  To  the  left  a  red  blood-corpuscle  with  basophilic,  karyochromatophilic 
granules.     Prototype  of  malarial  parasite.     On  the  right  a  red  blood -cor- 
puscle with  remains  of  nucleus. 

Sexual  Forms  or  Gametes. 

47.  An  earlier  quartan  gamete  (macrogamete  in  sphere  form),  female. 

48.  An  earlier  quartan  gamete  (microgametocyte),  male. 

49.  Tertian  gamete,  male  form  (microgametocyte). 

50.  Tertian  gamete,  female  (macrogamete). 

51.  Tertian  gamete  (microgametocyte)  still  within  a  red  blood-corpuscle. 

52.  Macrogamete  tertian  within  a  red  blood-corpuscle. 

53.  Tropical  fever,     (^stivo-autumnal)  gamete,  half  moon  (crescent)  still 
lying  in  a  red  blood-corpuscle.     In  the  middle  is  the  pigment.     The  concave 
side  of  the  crescent  is  spanned  by  the  border  of  the  red  blood-corpuscle. 

54.  Gamete,  tropical  fever  parasite. 

55.  Gamete  of  tropical     fever  parasite  heavily  pigmented. 

56.  Gamete  of  the  tropical  fever  parasite  (flagellated  form),  microgameto- 
cyte sending  out  microgametes  (flagella  or  spermatozoa). 


PLATE  IV. 


MALARIA.  191 

thence,  by  way  of  the  veneno-salivary  duct  in  the  hypopharynx,  they  are 
introduced  into  the  circulation  of  the  person  bitten  by  the  mosquito, 
and  start  a  nonsexual  cycle.  As  the  sexual  life  takes  place  in  the 
mosquito,  this  insect  is  the  definitive  host — man  is  only  the  interme- 
diary host. 

There  are  three  species  of  malarial  parasites:  (i)  the  Plasmodium 
vivax,  that  of  benign  tertian — cycle,  forty-eight  hours;  (2)  the  Plasmo- 
dium malariae,  that  of  quartan — cycle,  seventy-two  hours;  and  (3)  the 
Plasmodium  falciparum,  that  of  aestivo-autumnal  or  malignant  tertian- 
cycle  of  forty-eight  hours. 

Variations  in  cycles  may  be  produced  by  infected  mosquitoes  biting 
on  successive  nights,  so  that  one  crop  will  mature  and  sporulate  twenty- 
four  hours  before  the  second.  This  would  give  a  quotidian  type  of 
fever.  In  aestivo-autumnal  infections  anticipation  and  retardation  in 
the  sporulation  causes  a  very  protracted  paroxysm,  lasting  eighteen  to 
thirty-six  hours;  this  tends  to  give  a  continued  fever  instead  of  the 
characteristic  type. 

UNSTAINED  SPECIMEN  (FRESH  BLOOD). 


P.  vivax. 
(Benign  tertian.) 

P.  malariae. 
(Quartan.) 

P.  falciparum. 
(Malignant  tertian) 
(iEstivo-autumnal.) 

Character  of  the    Swollen    and    light    About  the  size  and 

Tendency  to  distor- 

infected   red     :       in  color. 

color  of  a  normal 

tion   of   red   cell 

cell. 

red  cell. 

rather  than  cre- 

. 

nation.  Shriveled 

appearance. 

(Brassy  color.) 

Character    of          Amoeboid     outline.   Has  band  or  ribbon- 

Small,    distinc  1  1  y 

young  schizont        Rarely  more  than        like   appearance. 

round,  crater  like 

one  in  r.c. 

dots     not     more 

than       one-sixth 

diameter   of    red 

cell.  Two  to  foui 

parasites   in   one 

red  cell  common. 

Amoeboid  with  fine    Rather     oval     in 

Only  seen  in  over- 

Character   of   ma-       yellow-biown  pig-        shape.      Sluggish 

whelming    infec- 

ture schizont           ment  which  shows        movement  of  per- 

tion.  Have  scanty 

active    swarming        ipherally    placed 

fine    black    pig- 

movement. 

coarse  black  pig- 

ment      clumped 

ment. 

together. 

192 


THE  PROTOZOA. 
STAINED  SPECIMEN. 


P.  vivax. 
(Benign  tertian  ) 


P.  malariae. 
(Quartan.) 


P.  falciparum. 
(Malignant  tertian) 
ti  vo-autumnal . ) 


Character   of    in- 

Larger and  lighter    About   normal   size    Shows        distortion 

fected  red  cell. 

pink  than  normal        and  staining.                and    some   poly- 

red    cell.    Shows 

chromatophilia 

"Schiiffner's 

and    stip  p  1  i  n  g. 

dots." 

Rarely    we    have 

coarse     cleft-like 

reddish       dots  — 

Maurer's  spots. 

Character    of 

Chromatin        mass 

Round    rings    with 

Very    small    sharp 

young  schizont. 

usually  single  and 

the        chromatin        band-like     rings, 

situated       inside 

mass  almost  cen-        with  a  chromatin 

the    ring    of   the 

trally  situated. 

mass    piotruding 

irregularly     out  - 

outside  the  ring. 

lined    blue  para- 

Often appears  on 

site. 

periphery  of  red 

cell.     Frequently 

two       chromatin 

dots. 

Character  of  ma- 

Fine pigment  rather 

Coarse     pigment    Very  rarely  seen  in 

ture    schizont 

evenly     distribu- 

rather   peripher-        peripheral   circu- 

ted  in  irregularly 

ally   arranged  in        lation  in  ordinary 

outlined  parasite. 

an  oval  parasite.           infection.     P  i  g  - 

ment      clumps 

Character  of  me-    Irregular      division 

Rather  regular  di-         early. 

rocyte. 

into     fifteen     or 

vision   into   8   or    Sporulation    occurs 

more      spore-like 

10    merozoites  —        in  spleen,  brain, 

chromatin        dot 

Daisy. 

etc.      Rarely     in 

segments. 

peripheral    circu- 

lation.   8    to    10 

chromatin    stain- 

ing  merozoites. 

Character  of  gam- 

Round. 

Round. 

Crescent. 

etes. 

In  the  diagnosis  of  malaria  one  should  always  examine  both  a  fresh 
specimen  and  a  stained  one,  as  each  method  gives  valuable  information 
in  differentiating  species.  When  time  will  not  permit  the  examination 
by  both  methods,  always  use  the  smear  stained  by  Wright's  stain,  as 
the  small  peripherally  situated  rings  of  aestivo-autumnal  fever  may 
escape  notice  in  a  fresh  specimen. 

SARCOSPORIDIA. 
Sarcosporidia  are  sporozoa  found  in  the  striped  muscles  of  various 


SARCOSPORIDIA. 


193 


mammals  and  birds.  They  are  common  in  the  pig  and  mouse  and 
have  been  reported  for  man. 

They  are  known  also  as  Miescher's  tubes  when  in  muscle  fibers. 
They  are  divided  into  three  genera:  Miescheria,  when  parasitic  in 
muscle  fiber;  Balbiania,  when  parasitic  in  the  intervening  connective 
tissue  of  the  muscles,  and  Sarcocystis. 

In  addition  to  the  protozoa  previously  referred  to,  there  are  certain 
infections  which  are  considered  by  certain  authors  to  be  protozoal  in 
nature. 


FIG.  60. — Diagram  showing  development  of  different  species  of  malarial  parasites. 

Cytorrhyctes  vaccinae. — These  parasites  develop  within  the 
epithelial  cells  of  stratified  epithelium.  In  vaccinia,  Councilman  and 
his  colleagues  consider  that  the  development  only  takes  place  in  the 
cytoplasm  of  the  cell.  In  variola,  however,  the  developmental  cycle 
affects  the  nucleus. 

Cytorrhyctes  luis,  reported  as  the  cause  of  syphilis,  sporulates  in  the 
blood-vessels  and  in  the  connective  tissue,  not  in  epithelial  cells. 

Cytorrhyctes  scarlatinae  was  reported  by  Mallory  to  have  been 
found  in  the  skin  in  four  cases  of  scarlet  fever. 


CHAPTER  XVII. 
FLAT  WORMS. 


CLASSIFICATION  or  THE  PLATYHELMINTHES  (FLAT  WORMS). 


Class 


Trematoda 


Family 


Fasciolklae 


0 


Cestoda 


Paramphistomidae 

\r^\ 
Schislocomklae 

Dibothriocephalidae 


Taeniidae 


Genus 

Species 

Fasciola 

F.  hepatica 

Fasciolopsis 

F.  buski 

Dicrocoelium 

D.  lanceatum 

Pajagonimus 

P.  westermani 

Qpisthorchis 

O.  felineus 

Clonorchis 

G.  sinensis 
,  C.  endemicus 

V 

Heterophyes 
Cladorchis 

H.  heterophyes 
C.  watsoni 

\  Gastrodiscus 

G   hominis 

'  S.  haematobium 

Schistosomum 

S.  japonicum 

S.  mansoni 

ae       Dibothriocephalus 

D.  latus 

f  Dipylidium 

D.  caninum 

Hymeode.pis 

H.  nana 
H.  diminuta 

T 

'  T.  solium 

1  asnia 

T.  saginata 

Davainea 

D.  madagascanensis. 

NOTE. — Two  larval  Taeniidae  are  found  in  man  (Cysticercus  cellulosae  and 
Echinococcus  polymorphus) . 

Also  two  larval  Dibothriocephalidae  (Sparganum  mansoni  and  Sparganum 
prolifer). 

TREMATODES  OR  FLUKES. 

Flukes  are  generally  leaf-like  in  outline,  exhibiting  marked  variation 
in  size  and  shape.  The  largest  human  fluke,  F.  buski,  is  from  2  to  3 
inches  in  length,  while  the  H.  heterophyes  is  less  than  1/12  of  an  inch 
in  length.  The  most  important  fluke,  the  liver  fluke,  Clonorchis 
endemicus,  is  flat  and  almost  transparent,  while  the  almost  equally 
important  lung  fluke,  the  Paragonimus  westermani,  is  oval,  almost 
round  and  reddish-brown  in  color.  With  the  exception  of  the  Schis- 
tosomidce,  all  flukes  are  hermaphrodites,  and,  with  the  exception  of  this 

194 


DISTOMIASIS.  195 

family,  all  flukes  have  operculated  eggs.  The  only  other  operculated 
(with  a  lid)  eggs  we  meet  with  in  man  are  those  of  the  Dibothrioceph- 
alidae. 

Flukes  have  two  suckers  which,  except  in  the  Paramphistomidae, 
are  quite  near  each  other — one  is  termed  the  oral  sucker  and  the 
other  the  ventral  sucker  or  acetabulum.  The  intestinal  tract  consists 
of  a  pharynx,  proceeding  from  the  oral  sucker,  which  bifurcates  and 
terminates  in  blind  intestinal  caeca.  The  life  history  of  the  important 
human  flukes  is  unknown.  It  is  supposed  that  this,  in  a  measure,  may 
resemble  that  of  the  common  liver-fluke  disease  of  sheep  (sheep  rot). 
In  this  the  eggs  containing  a  ciliated  embryo  (Miracidium)  pass  out  in 
the  faeces.  This  embryo  is  hatched  out  and,  gaining  the  water,  swims 
about  actively  until  it  reaches  some  suitable  mollusk  or  crustacean 
(Limnaea  trunca'tula).  By  means  of  a  pointed  end,  it  bores  its  way 
into  the  body  of  the  gastropod  and  becomes  either  a  bag-like  structure 
(the  sporocyst)  or  develops  into  a  creature  with  an  alimentary  canal 
(redia).  From  the  sporocyst  or  redia  minute  little  worms  resembling 
adult  flukes  in  possessing  suckers,  but  differing  in  the  possession  of  a 
tail,  develop  (Cercaria).  Having  reached  maturity,  these  cercariae 
leave  the  sporocyst  or  redia,  and,  as  in  case  of  F.  hepatica,  become 
encysted  on  blades  of  grass,  to  be  eaten  by  sheep  and  again  com- 
mence the  cycle.  The  encysted  cercariae  develop  into  adult  liver 
flukes.  It  is  probable  that  with  many  flukes  the  cercariae  enter  some 
host,  as  mollusk,  insect  or  fish,  and  that  it  is  by  eating  such  animals  as 
food  that  man  becomes  infected.  Looss  thinks  it  possible  that  the 
miracidium  of  Schistosum  haematobium  may  bore  its  way  directly  into 
man,  as  do  the  larvae  of  the  hook-worm.  Manson  also  suggests  that 
the  reporting  by  Musgrave  of  100  mature  lung  flukes  in  a  psoas 
abscess  makes  it  very  probable  that  these  parasites  entered  the  body 
as  miracidia.  The  idea  in  China  is  that  the  infection  with  the  com- 
mon liver  fluke  of  man  is  brought  about  by  eating  fish.  Fluke  disease 
is  generally  known  as  distomatosis  or  distomiasis. 

LIVER  FLUKES. 

Fasciola  hepatica. — This  fluke,  while  of  enormous  economic 
importance  by  reason  of  destruction  of  sheep,  has  only  been  reported 


196 


FLAT   WORMS. 


23  times  in  man,  and  in  these  instances  does  not  seem  to  have  occa- 
sioned marked  symptoms.  There  is,  however,  a  possible  importance 
of  F.  hepatica  in  connection  with  a  peculiar  affection  known  as 
"halzoun."  This  results  from  the  eating  of  raw  goats'  liver,  and  it  is 
supposed  that  the  'flukes  crawl  up  from  the  stomach  and,  entering  the 


5. 


FIG.  61. — Trematodes  of  man,  natural  size,  i,  Oonorchis  endemicus  (Opisthor- 
chis  sinensis) ;  2,  Gastrodiscus  hominis;  3,  Dicrocoelium  lanceatum;  4,  Hetcrophyes 
heterophyes;  5,  Schistosomum  haematobium;  6,  Fasciola  hepatica;  7,  Paragonimus 
westermani;  8,  Fasciolopsis  buski;  9,  Opisthorchis  felineus;  10,  anatomy  of  C.  en- 
demicus (enlarged).  G.  P.,  genital  pore;  V.  S.,  ventral  sucker;  V.  G.  vitelline 
glands;  R.  S.,  receptaculum  seminis;  T.,  branched  testicles. 

larynx  or  attaching  themselves  about  the  glottis,  produce  the  asphyxia 
characteristic  of  the  disease. 

Dicrocoelium  lanceatum. — This  has  only  been  reported  7  times 
in  man.  The  symptoms  are  unimportant;  the  fluke  is  about  1/3  of  an 
inch  long. 

Clonorchis  endemicus. — This  fluke  and  the  C.  sinensis  are  the 
most  important  of  the  human  liver  flukes.  Until  recently  these  flukes 


DISTOMIASIS.  197 

were  known  as  Opisthorcis  sinensis.  This  fluke  is  very  common  in 
China  and  Japan — in  certain  sections  of  Japan,  20%  of  the  population 
being  infected.  This  fluke  is  about  3/4  of  an  inch  long  and  1/6  of  an 
inch  broad.  When  squeezed  out  of  the  thickened  bile  ducts  it  is  so 
transparent  and  glairy  as  almost  to  resemble  glairy  mucus.  This 
fluke  is  supposed  to  produce  most  serious  symptoms,  as  indigestion, 
swelling  and  tenderness  of  liver,  ascites,  oedema,  and  a  fatal  cachexia. 
As  a  matter  of  fact,  many  physicians  in  China  attribute  very  little 
pathogenic  importance  to  it.  The  disease  is  diagnosed  by  the  presence 
of  the  ova  in  the  stools. 

Opisthorchis  felineus. — This  fluke  is  smaller  than  the  C.  endemi- 
cus,  and  is  a  common  parasite  of  the  gall  bladder  and  bile  ducts  of  cats. 
In  certain  parts  of  Siberia  the  parasite  is  found  in  more  than  6%  of  the 
human  autopsies. 

Intestinal  Flukes. 

Cladorchis  watsoni. — This  fluke  is  about  1/3  of  an  inch  long,  and 
has  an  indistinct  oral  sucker  and  a  large  sucker  at  the  other  end. 
This  parasite  has  only  been  once  reported. 

Gastrodiscus  hominis. — This  fluke  is  about  1/4  of  an  inch  long 
and  has  a  disk  about  1/6  of  an  inch  in  diameter  from  which  proceeds  a 
teat-like  projection,  bearing  an  oral  sucker.  While  it  has  only  been 
reported  twice  for  man,  indications  are  that  it  is  probably  fairly 
common  in  India  and  Assam. 

Fasciolopsis  buski. — This  is  probably  a  rather  common  parasite 
in  India,  as  Dobson  found  the  eggs  in  i%  of  the  stools  of  more  than  1000 
coolies.  The  fluke  is  from  2  to  3  inches  in  length  and  about  1/2  of  an 
inch  in  breadth.  It  is  thick,  brown  in  color  and  has  a  very  large 
acetabulum.  These  parasites  cause  dyspeptic  symptoms  and  an 
irregular  diarrhcea. 

Heterophyes  heterophyes. — This  exceedingly  small  fluke,  which 
can  be  recognized  by  its  small  size  (less  than  1/12  of  an  inch  long)  and 
large,  prominent  acetabulum,  was  formerly  supposed  to  be  rare.  Looss, 
however,  has  shown  that  it  is  quite  common  in  Egypt,  he  having  found 
it  twice  in  Alexandria  in  9  autopsies.  The  parasites  occupy  the  ileum. 
It  is  common  in  dogs. 


Tp8  FLAT    WORMS. 

LUNG  FLUKES. 

Paragonimus  westermani. — In  certain  parts  of  Japan  and  For- 
mosa it  is  estimated  that  as  many  as  10%  of  the  inhabitants  may  harbor 
this  parasite. 

It  is  also  common  in  China,  and  recently  many  cases  have  been 
reported  in  the  Philippines.  Dr.  Stiles  states  that  around  Cincinnati 
there  is  quite  a  heavy  infection  among  the  hogs,  so  that  it  may  be  that 
certain  cases  diagnosed  in  man  as  pulmonary  tuberculosis  may  be  due 
to  this  disease. 

It  is  popularly  known  as  endemic  haemoptysis  on  account  of  the 
accompanying  symptoms  of  chronic  cough  and  expectoration  of  a 
rusty-brown  sputum.  After  violent  exertion,  and  at  times  without 
manifest  reason,  attacks  of  haemoptysis  of  varying  degrees  of  severity 
come  on.  The  characteristic  ova  are  constant  in  the  sputum  and 
establish  the  diagnosis.  The  fluke  itself  is  a  little  more  than  1/3  of  an 
inch  long  and  is  almost  round  on  transverse  section.  It  is  rather  flesh- 
like  in  appearance.  The  flukes  are  usually  found  in  tunnels  in  the 
lungs,  the  walls  of  which  are  of  thickened  connective  tissue.  There 
may  be  also  cysts  formed  from  the  breaking  down  of  adjacent  tunnel 
walls.  In  addition  to  lung  infection  with  this  fluke,  brain,  liver  and 
intestinal  infections  may  be  found.  Musgrave  was  the  first  one  to  call 
attention  to  the  frequency  of  general  infection  with  this  parasite 
(paragonomiasis)  in  the  Philippines.  He  found  it  in  1 7  cases  in  one  year. 
The  life  history,  beyond  the  stage  of  miracidium,  is  unknown.  As 
stated  previously,  Manson  suggests  that  the  miracidium  may  enter 
directly  by  the  skin. 

BLOOD  FLUKES. 

Schistosomun  haematobium. — Flukes  of  the  circulatory  system. 
These  flukes  are  of  great  importance  in  Egypt,  Japan  and  the  West 
Indies.  The  disease  is  named  bilharziasis  after  Bilharz  who  first  as- 
sociated the  parasite  and  the  disease.  It  seems  probable  that  there  are 
at  least  three  human  species,  differentiated  principally  by  the  character 
of  the  egg.  In  the  blood-fluke  disease  of  Egypt,  S.  haematobium,  the 
parasite  chiefly  infects  the  bladder  and  the  egg  has  a  terminal  spine. 


BILHARZIASIS.  1 99 

The  lateral-spined  ovum  is  also  found  in  the  faeces.  In  the  West 
Indies,  as  shown  by  the  reports  of  Surgeon  Holcomb  from  Porto  Rico, 
rectal  bilharziasis  is  rather  common.  In  these  cases  the  egg  is  prac- 
tically always  lateral-spined.  The  adults  of  this  species,  the  S. 
mansoni,  are  scarcely,  if  at  all,  to  be  distinguished  from  the  S.  haema- 
tobium.  With  the  S.  japonicum,  the  name  of  the  Eastern  species,  there 
is  not  only  the  difference  in  that  the  eggs  are  without  spines,  but,  in 
addition,  the  skin  of  the  adult  parasite  is  nottuberculated,as  is  the  case 
with  the  other  two  species.  Catto  considers  that  the  S.  japonicum  may 
live  in  both  arteries  and  veins.  The  other  two  species  only  live  in 
branches  of  the  portal  vein.  The  blood  flukes  are  about  1/2  inch 
long.  All  of  these  flukes  are  hermaphroditic,  but  live  separately  until 
maturity.  At  this  time  the  female  enters  what  is  known  as  the  gynaeco- 
phoric  canal  of  the  male;  this  canal  is  formed  by  the  infolding  of  the 
sides  of  the  flat  male  fluke,  thus  giving  a  rounded  appearance  to  the 
male.  The  female  is  longer  than  the  male  (about  5/6  of  an  inch  long), 
and  is  thread-like.  Her  two  extremities  project  from  the  canal  of  the 
male  in  which  she  lives. 

The  most  prominent  symptoms  of  the  Bilharz  disease  are  haema- 
turia  and  bladder  irritation;  later  on  calculus  formation.  In  rectal 
bilharziasis  the  symptoms  are  more  those  of  bleeding  piles  or  of  a  mild 
dysentery.  In  the  Japanese  infection  the  symptoms  point  more  to 
liver  and  spleen,  there  being  ascites,  cachexia  and  a  bloody  diarrhoea. 

The  life  history  is  not  known  of  any  of  these  flukes.  Looss  con- 
jectures that  it  is  probable  that  the  miracidium  enters  the  skin,  not  re- 
quiring an  intermediary  host.  Frequent  experiments  have  failed  to 
show  any  mollusk,  etc.,  which  attracted  the  embryo.  Evidence  seems 
to  show  that  those  who  are  constantly  wading  about  in  the  water 
of  the  pools  or  the  mud  of  the  fields  are  the  ones  most  subject  to 
infection. 

If  urine  containing  eggs  is  diluted  with  water  the  miracidium 
breaks  out  of  the  shell  and  swims  about  as  if  in  search  of  some 
desired  object. 

The  views  are  also  entertained  that  the  miracidium  may  gain  access 
to  the  body  through  the  drinking  water;  there  is  much  evidence  against 
this.  However  access  to  the  body  is  gained,  it  is  known  that  the  larval 


200  FLAT    WORMS. 

forms  make  their  way  to  the  liver  where  they  develop.  Arriving  at 
maturity,  the  males  and  females  become  united  and  proceed  to  the 
terminal  branches  of  the  portal  vein^  where  the  irritating  eggs,  given  off 
by  the  female,  give  rise  to  the  symptoms. 

CESTODE  OR  TAPE- WORM  INFECTIONS. 

The  cestodes  and  trematodes  constitute  the  two  great  divisions  of 
the  flat-worms.  Anatomically,  a  tape-worm  may  be  considered  as  a 
series  of  individual  flukes  united  in  one  ribbon-like  colony.  The 
cestode  segments,  or  proglottides,  differ,  however,  from  the  flukes  in  not 
having  an  alimentary  canal  and  in  having  a  cellular  instead  of  a 
chitinous  external  covering. 

A  tape-worm  is  divided,  into  the  segment-producing  controlling 
head  and  the  series  of  segments  or  proglottides  together  known  as  the 
strobila.  The  head  and  neck  together  form  the  scolex.  Tape-worm 
heads  are  provided  with  suctorial  or  hook-like  suckers,  or  both,  to 
enable  them  to  hold  on  to  the  intestinal  mucosa.  The  importance  of 
the  head  is  generally  recognized  by  the  well-known  fact  that  the  per- 
manent evacuation  of  one  of  these  parasites  is  only  arrived  at  when  the 
head  as  well  as  the  segments  is  expelled.  Otherwise,  additional 
segments  will  be  produced.  Even  in  tape-worms  twenty-five  to 
thirty  feet  in  length,  the  head  is  no  larger  than  a  small  shot.  It 
carries  the  suckers  or  booklets  which  best  enable  us  to  differentiate 
the  different  species.  The  segments  adjacent  to  the  head  are  im- 
mature— the  sexually -mature  ones  being  found  from  the  middle  of  the 
body  onward.  The  sexually-mature  segment  possesses  a  varying 
number  of  testicles:  3  in  H.  nana  and  as  many  as  2000  in  T.  saginata. 
As  with  the  flukes,  they  also  have  ovaries,  yolk  glands,  uterus,  genital 
pore,  etc.  The  location  of  the  genital  pore  and  the  character  of  the 
branching  of  the  uterus  are  of  the  greatest  importance  in  differentia- 
tion. The  sexually-mature  proglottides  may  either  expe  their  ova, 
when  these  would  be  found  in  the  faeces  or,  as  is  common,  they  break  off 
and  pass  out  themselves  in  the  faeces  They  then  either  expel  the  eggs 
or  may  be  eaten  by  some  animal  and  in  this  way  effect  an  entrance  for 
their  ova.  The  "hexacanth"  or  6-hooked  embryo,  also  called  the 
onchosphere,  is  the  essential  part  of  the  egg.  The  egg  shell  is  dis- 


*  TAPE   WORMS. 


201 


solved  off  in  the  alimentary  canal  of  the  animal  ingesting  it,  and  the 
onchosphere  bores  its  way  through  the  gut  to  later  become  encysted  in 
various  tissues.  In  some  tape-worms  a  ciliated  embryo  is  liberated 
from  the  egg  shell  and  swims  about  actively  to  enter  some  fish  or  other 
animal.  When  the  6-hooked  embryo  reaches  its  proper  tissue,  the 
hooklets  are  discarded  and  a  scolex  similar  to  the  parent  one  is  devel- 


to. 


C. 


FIG.  62. — Tape  worms.  A.  i,  2  and  3,  Scolex,  proglottides  and  ovum  of  Taenia 
solium;  B.  4,  5,  6  and  7,  Scolex,  proglottides  and  ovum  of  Dibothriocephalus 
latus;  C,  8,  9  and  10,  Scolex,  proglottides  and  ovum  of  Taeenia  saginata. 


oped.  At  this  time  we  have  a  bladderlike  structure  with  the  scolex 
inverted  in  it.  This  little  cyst  with  its  scolex  when  ingested  by  another 
animal  is  digested,  and  the  scolex  establishing  itself  in  the  intestine, 
develops  a  series  of  segments.  The  ciliated  embryo  of  the  D.  latus 
does  not  form  a  cyst,  but  instead  a  worm-like  creature  similar  to  the 
adult.  This  is  termed  a  Plerocercoid.  Small  laminated  calcareous 
corpuscles  are  characteristic  of  cestode  tissue. 
14 


202  FLAT    WORMS. 


INFECTIONS. 

Taenia  saginata.  —  This  very  widely  distributed  tape-worm  is  often 
termed  the  unarmed  tape-worm,  to  distinguish  it  from  the  T.  solium  or 
armed  tape-worm.  It  is  from  10  to  25  feet  long  and  has  several  hun- 
dred proglottides.  The  small  pear-shaped  head  has  4  pigmented 
elliptical  suckers  and  no  hooklets.  The  segments  are  plumper  than 
those  of  T.  solium,  hence  the  name  saginata.  The  single  lateral 
genital  pore  projects  markedly  and  in  a  series  of  segments  presents,  as 
a  rule,  first  on  one  side,  and  then  on  the  opposite  side  of  the  next  seg- 
ment (alternating).  The  best  way  to  distinguish  a  segment  of  the  T. 
saginata  from  the  T.  solium  is  by  counting  the  number  of  lateral 
uterine  branches;  these  number  15  to  30  and  branch  dichotomously. 
The  lateral  divisions  of  the  uterus  of  the  T.  solium  are  tree-like  in  their 
blanching  and  only  number  5  to  12  on  each  side.  The  ox  is  the  in- 
termediate host.  The  6-hooked  embryo,  having  worked  its  way  from 
the  alimentary  canal  to  the  muscles  or  liver  of  the  ox,  become  encysted 
(Cysticercus  bovis).  This  little  bladder-like  structure  is  about  1/4  by 
i  /  3  inches.  Being  ingested  by  man's  eating  raw  or  imperfectly  cooked 
meat,  the  adult  stage  becomes  established  in  his  alimentary  canal. 
In  Abyssinia  the  infection  is  said  to  be  universal,  and  a  man  without  a 
tape  worm  to  be  a  freak.  An  important  point  is  the  fact  that  the 
larval  stage  never  appears  in  man.  It  is  this  fact  which  makes  it  a  so 
much  less  dangerous  parasite  than  the  T.  solium,  which  readily  es- 
tablishes a  larval  existence  in  man  if  the  ova  are  introduced  into  the 
human  stomach.  Cooking  meat  always  destroys  the  Cysticercus. 
A  period  of  about  2  months  elapses  after  the  ingestion  of  the  Cysticer- 
cus before  the  mature  segments  pass  out  of  the  rectum.  These  not 
only  make  their  exit  with  the  faeces,  but  are  also  capable  of  wandering 
out  at  other  times.  In  this  they  differ  from  the  segments  of  T.  solium. 
T.  saginata  next  to  H.  nana  is  the  common  tape-worm  of  the  United 
States.  Dr.  Stiles  has  examined  several  hundred  tape-worms  during 
the  past  few  years  and  has  found  only  one  T.  solium. 

Taenia  solium.  —  The  measly  -pork  tape-worm  is  smaller  than  the 
T.  saginata  and  differs  from  it  in  having  a  globular  head,  with  a  rostel- 
lum,  which  is  crowned  by  26  to  28  hooklets.  The  segments  have 
only  5  to  10  branches  and  are  expelled  only  at  the  time  of  defecation. 


IAIM.  WORMS.  203 

The  segments  or  the  ova  having  been  ingested  by  a  hog,  the  6-hooked 
embryo  is  liberated  and  becomes  encysted  in  the  muscles  of  the  hog,  as 
an  invaginated  scolex.  Pork  containing  this  Cysticercus  (Cysticercus 
cellulosae)  is  known  as  measly  pork.  If  one  by  chance  should  carry 
the  eggs  on  his  fingers  to  his  mouth,  as  the  result  of  examining  mature 
segments,  the  larval  stage  may  be  established  in  man.  If  this  infection 
is  not  heavy,  very  few  symptoms  may  be  observed.  The  Cysticercus, 
however,  tends  to  invade  the  brain,  next  in  frequency  the  eye,  and  so 
causes  convulsions,  death  or  blindness.  Instead  of  only  being  the 
size  of  a  pea,  these  cysts  when  forming  in  the  brain  may  be  the  size  of  a 
walnut  or  larger.  T.  solium  is  comparatively  common  in  North 
Germany,  but  is  exceedingly  rare  in  England  and  the  United  States. 

Taenia  africana. — This  is  an  unarmed  tape-worm,  only  about  5 
feet  long.     It  was  found  in  a  native  soldier  in  German  East  Africa. 

Hymenolepis  nana. — This  is  generally  known  as  the  dwarf  tape 
worm — it  is  the  smallest  of  the  human  tape-worms.  It  is  from  1/4  of  an 
inch  to  1/2  inch  in  length,  and  is  less  than  1/25  of  an  inch  in  breadth. 
The  genus  Hymenolepis  has  lateral  g.  pores,  all  of  which  are  on  the 
same  side.  The  head  has  4  suckers  and  a  rostellum,  which  is  usually 
invaginated.  The  rostellum  has  24  to  30  booklets  encircling  it.  The 
eggs  of  this  species  are  quite  characteristic,  there  being  2  distinct 
membranes.  The  inner  one  has  2  distinct  knobs,  from  which  thread- 
like filaments  proceed.  The  eggs  of  the  H.  diminuta  have  a  thicker, 
striated  outer  membrane  and  there  are  no  filaments.  The  eggs  of  the 
Dipylidium  caninum  are  similar,  but  are  found  in  the  faeces  in  aggre- 
gations— several  eggs  in  a  packet.  The  dwarf  tape-worm  has  been 
found  to  be  the  most  common  tape-worm  in  the  United  States.  Dr. 
Stiles  found  it  in  about  5%  of  children  in  a  Washington  orphanage. 
It  has  been  estimated  that  in  certain  parts  of  Italy  10%  of  the 
children  may  be  infected.  The  symptoms,  especially  nervous  ones, 
may  be  marked  in  this  infection.  Although  very  small,  yet  the 
number  of  parasites  may  be  very  great,  even  more  than  1000.  A 
form  found  in  rats,  which  may  be  identical  with  H.  nana,  does  not 
require  an  intermediate  host.  The  6-hooked  embryo  bores  into 
the  intestinal  villus  and  there  delveops  a  Cercocystis  (larva  of 
small  dimensions  with  but  little  fluid).  When  fully  developed,  it 


204  FLAT    WORMS. 

drops  into  the  lumen  of  the  gut,  and  a  new  parasite  is  added  to  the 
already  existing  number  of  parasites.  This  explains  the  heavy 
infection.  H.  diminuta  and  H.  lanceolata  have  also  been  reported 
for  man  a  few  times. 

Dipylidium  caninum. — This  is  a  common  parasite  of  dogs  and 
cats.  The  larval  stage  is  passed  in  lice  and  fleas.  The  cases  of  human 
infection  have  been  principally  in  children,  probably  from  getting  dog 
lice  or  fleas  in  their  mouths.  The  head  has  4  suckers  and  a  rostellum. 
which  has  3  or  4  rows  of  encircling  hooklets.  The  segments  have  the 
shape  of  melon  seeds  and  have  bilateral  genital  pores. 

Davainea  madagascariensis. — This  tape-worm  has  been  found  in 
Siam  and  Mauritius.  It  is  about  10  inches  long.  The  head  has  4 
suckers  and  a  rostellum  with  90  hooklets.  The  genital  pores  are 
unilateral.  The  cockroach  is  supposed  to  be  the  intermediate  host. 

DIBOTHRIOCEPHALID.E  INFECTIONS. 

Dibothriocephalus  latus. — This  is  frequently  termed  the  broad 
Russian  tape-worm.  It  has  a  small  olive-shaped  head  with  2  deep 
winding  suctorial  grooves  on  each  side;  it  has  neither  rostellum  nor 
hooklets.  The  segments  are  quite  broad,  being  about.  1/2  by  1/5  in. 
At  the  end  of  the  strobile  they  are  more  nearly  square.  The  segments 
are  very  numerous,  3000  or  more.  The  fully  developed  worm  is  about 
30  feet  long.  The  uterus  in  each  segment  is  rosette-shaped  and  the 
genital  pore  is  ventrally  situated.  The  eggs  of  this  species  have  an 
operculum  and  a  ciliated  embryo.  This  ciliated  embryo  swims 
around  and  either  enters  some  fish,  especially  pike,  directly  or  through 
an  as  yet  unknown  intermediary.  This  parasite  produces  an  intense 
anaemia  similar  to  pernicious  anaemia.  It  is  a  frequent  parasite  in 
Switzerland,  Bavaria,  Japan,  Scandinavia  and  Russia.  Recently 
several  cases  have  been  reported  from  our  Northwest,  and  some  of  the 
fish  of  the  waters  of  that  region  are  said  to  be  infected.  The  larva  is  a 
plerocercoid  and  is  about  i  inch  long.  It  is  said  that  salting,  smoking 
or  other  ordinary  methods  of  preserving  fish  will  not  kill  it. 

A  tape-worm,  Diplogonoporus  grandis  has  been  reported  from 
Japan.  In  this  there  are  2  complete  sets  of  genital  organs  to  each 
segment. 


HYDATID   DISEASE. 


205 


SOMATIC   T^NIASIS. 

While  rarely  we  may  have  the  larval  stage  of  T.  solium  present  in 
man,  and  while  certain  bothriocephalid  larvae  (Sparagnum  mansoni 
and  Sparganum  proliferum)  infect  man,  yet  they  are  unimportant  as 
compared  with  the  larval  stage  of  the  Taenia  echinococcus.  The 
adult  stage  of  this  parasite  is  passed  in  dogs.  It  is  one  of  the  smallest 
tape-worms  known,  being  only  about  1/6  in.  long.  It  has  a  head  with  4 


FIG,  63. — Tape  worms,  i,  2  and  3,  head,  melon-shaped  segments  and  egg 
packet  of  Dipylidium  caninum;  4,  5,  6  and  10,  entire  worm  magnified,  head,  larval 
stage  in  intestinal  villus  and  ovum  of  Hymenolepis  nana;  7,  echinococcus  cyst; 
A,  mother  cyst;  D,  daughter  cyst;  E,  granddaughter  cyst;  C,  scolex  in  brood 
capsule;  B,  brood  capsule;  G,  parenchymatous  layer;  F,  laminated  layer;  8  and  9, 
Taenia  echinococcus;  9,  natural  size. 

suckers  and  a  rostellum  encircled  with  hooks.  There  are  only  3  to  4 
segments.  The  larval  stage,  on  the  contrary,  gives  oneof  the  largest  of 
larval  cestodes.  The  larval  stage  is  also  found  in  hogs  and  sheep,  and 
it  is  probably  by  reason  of  the  dog  eating  the  echinococcus  cyst  of  such 
animals  at  the  abattoir  that  we  owe  the  increase  in  this  serious  infection. 


206  FLAT    WORMS. 

Man  contracts  the  infection  from  association  with  dogs.  The 
disease  is  peculiarly  prevalent  in  Iceland.  As  stated  above,  the  adult 
stage  is  passed  in  the  intestine  of  the  dog.  Should  the  egg-bearing 
segments  passed  by  the  dog  contaminate  the  hands  of  man  and  a  single 
egg  be  ingested,  we  may  have  hundreds  of  Taenia  larvae  produced. 
The  6-hooked  embryo,  leaving  its  shell,  bores  its  way  through  the  walls 
of  the  alimentary  tract  and  especially  seeks  the  liver,  just  as  the 
onchosphere  of  the  T.  solium  seeks  the  brain  and  eye. 

In  the  development  of  the  cyst,  after  the  onchosphere  has  come  to 
rest  at  some  point  in  the  liver,  we  have  formed  at  first  an  indistinctly 
laminated  external  envelope  with  coarsely  granular  fluid  contents. 
Later  on  the  contents  become  transparent,  and  2  distinct  layers  can  be 
observed:  (i)  The  external,  markedly  laminated  one,  and  (2)  the 
internal  one,  made  up  of  small  cells  externally  and  large  cells  and  cal- 
careous corpuscles  internally.  This  internal  lining  membrane  is 
known  as  the  parenchymatous  or  germinal  layer.  When  the  external 
layer  is  incised  it  curls  up  by  reason  of  its  elasticity.  This  is  character- 
istic of  such  a  cyst.  In  addition,  we  have  an  enveloping  connective- 
tissue  capsule  formed  by  the  surrounding  liver  substance.  From  the 
germinal  layer  arise  the  brood  capsules  and  the  scolices.  In  these 
brood  capsules  we  have  the  cellular  layer  external — just  the  reverse  of 
the  mother  cyst.  Scolices  may  develop  either  on  the  outside  or  inside 
of  these  brood  capsules.  It  is  interesting  to  note  that  one  onchosphere 
may  develop  hundreds  of  scolices.  From  the  parenchymatous  layer 
of  the  mother  cyst,  daughter  cysts  are  formed;  these  have  an  external 
stratified  layer  and  an  internal  parenchymatous  one;  within  them  a 
varying  number  of  scolices  may  develop.  From  these  daughter  cysts, 
granddaughter  cysts  may  arise — all  within  the  mother  cyst- — and 
hence  are  termed  endogenous. 

At  times  the  daughter  cysts  work  their  way  external  to  the  mother 
cyst  and  proceed  to  develop  in  a  manner  similar  to  the  endogenous 
formation.  The  exogenous  development  is  rare  in  man,  but  common  in 
hogs.  Hydatids  containing  no  scolices  are  called  sterile.  These  cysts 
may  be  as  large  as  a  child's  head,  but  are  usually  smaller.  The  fluid  of 
these  cysts  contains  about  i%  of  NaCl,  also  a  trace  of  sugar;  in  addition 
there  is  a  toxin  which  produces  urticaria  and  acts  as  a  cardiac  depress- 


LARVAL   TAPE    WORM    INFECTIONS. 


20' 


ant.  If  any  quantity  should  escape  into  the  peritoneal  cavity  at 
operation,  it  may  cause  death.  Hydatids  develop  very  slowly,  and  the 
duration  of  the  disease  is  usually  from  2  to  8  years. 

Sparganum  mansoni. — This  is  a  larval  bothriocephalid  which  is 


FIG.  64. — Ova  of  intestinal  parasites  with  measurements  in  mikrons.  i  Fasciola 
hepatica  (150x80);  2,  Taenia  saginata  (25);  3,  Ascaris  lumbncoides  (65x50); 
4,  Fasciolopsis  buski  (125x75);  5,  Necator  americanus  (70x40);  6,  Dibothrio- 
cephalus  latus  (70x45);  7,  Schistosomum  haematobium  (100x50);  8,  Trichoceph- 
alus  trichiurus  (55x25);  9,  Strongyloides  stercoralis  (60x30);  10.  Schistosomum 
mansoni  (110x60);  n,  Hymenolepis  nana  (56x53);  12,  Echinorhynchus  gigas 
(100x80);  13  and  15,  Oxyuris  vermicularis  (50x20);  14,  Heterophyes  heterophyes 
(28x16);  16,  Clonorchis  endemicus  (28x16). 

about  5  to  10  inches  long  and  has  been  reported  10  times  in  Japan.     It 
has  been  found  in  various  parts  of  the  body. 

Sparganum  prolifer. — This  has  been  reported  from  Japan  as  a 
larval  form  in  the  subcutaneous  tissue.  Stiles  has  found  these  larval 
forms  in  skin  lesions  in  Florida.  They  show  themselves  as  bizarre 
grub-like  forms. 


CHAPTER  XVIII. 


THE  ROUND  WORMS. 

CLASSIFICATION  OF  THE  NEMATHELMINTHES  (ROUND  WORMS). 

Class. 


/O 


Nematoda, 


Family.                          Genus. 

Species. 

Angiostomidae,                Strongyloides, 

S.  stercoralis. 

(Dracunculus, 

D.  medinensis. 

F.  bancrofti. 

F.  loa. 

Filariidae,                      1   Filaria, 

F.  perstans. 

I 

F.  demarquayi. 

F.  ozzardi. 

F.  philippinensis 

Trichotrachelidae, 

Trichocephalus, 
Trichinella, 

T.  trichiurus. 
T.  spiralis. 

Eustrongylus, 

E.  gigas. 

Strongylidae, 

TrichostrongyluS; 
Agchylostoma, 

T.  instabilis. 
A.  duodenale. 

Necator, 

N.  americanus. 

Ascaridae, 

Ascaris, 

A.  lumbricoides. 

A.  canis. 

Oxyuris, 

O.  vermicularis. 

Gigantorhynchus 

G.  gigas. 

{Hirudo, 

H.  medicinalis. 

Limnatis, 

L.  nilotica. 

Haemadipsa, 

H.  ceylonica. 

Acanthocephala, 
Hirudinea, 


NOTE. — The  Strongyloides  stercoralis  was  formerly  described  under  two  desig- 
nations: (i)  Anguillula  intestinalis,  a  parasitic  generation  and  (2)  Anguillula  ster- 
coralis, a  free  living  generation. 

ROUND  WORMS  OR  NEMATODES. 

All  nematodes  are  covered  by  a  cuticle  which  varies  in  thickness. 
The  sexes  are,  as  a  rule,  separate.  The  male  can  usually  be  recognized 
by  its  small  size,  its  curved  or  curled  posterior  end,  and  at  times  exhib- 
iting an  umbrella -like  expansion — the  copula  tory  bursa.  The  spicules, 
chitinous  copulatory  structures,  may  be  observed  drawn  up  in  the 
worm  or  projected  out  of  the  cloaca. 

Certain  papillae  in  the  region  of  the  anus  are  valuable  in  differen- 
tiation. In  the  female  the  vulva  is  usually  situated  about  the  middle 
of  the  ventral  surface. 

208 


COCHIN-CHINA  DIARRHCEA.  2OQ 

ANGIOSTOMID^:. 

Strongyloides  stercoralis.  —  This  parasite  was  formerly  thought 
to  be  the  cause  of  Cochin-China  diarrhoea.  It  presents  two  genera- 
tions: i.  Parasitical  or  intestinal  form.  2.  The  free  living  or  fecal 
form. 

i.  The  intestinal  form  (also  known  as  Anguillula  intestinalis)  is 
represented  only  by  females.  These  are  about  1/12  of  an  inch  long 
and  reproduce  parthenogenetically.  The  embryos  escape  from  the 
eggs  while  still  in  the  intestine,  so  that  in  the  faeces  we  only  find  actively 
motile  embryos.  The  eggs,  which  are  strung  out  in  a  chain,  never 
appear  in  the  faeces  except  during  purgation.  As  they  greatly  re- 
semble hook-worm  eggs,  this  is  a  point  of  great  practical  importance. 
In  fresh  faeces  we  find  hook-worm  eggs  and  Strongyloides  embryos. 
If  the  temperature  is  low,  these  rhabditiform  embryos  develop  into 
filariform  embryos,  which  being  ingested  form  the  infecting  stage. 
If  the  temperature  is  warm,  25°  to  35°  C.,  these  embryos  develop  into: 

(2)  The  free  living  form.  In  this  we  have  males  and  females; 
these  copulate  and  we  have  produced  rhabditiform  larvae,  which  later 
change  to  filariform  ones.  These  being  ingested,  start  up  the  parasiti- 
cal generation.  The  embryos  are  rather  common  in  stools  in  the 
tropics.  These  embryos  have  pointed  tails  and  are  about  250  x  i3/£. 
They  have  a  double  cesophageal  bulb. 


This  family  is  of  the  greatest  importance  to  man.  It  is  also  one 
about  which  much  confusion  exists.as  to  the  adult  type;  hence  anyone 
finding  adult  filariae  should  fix  them  in  hot  5%  glycerin  alcohol  (alcohol 
70%),  and  subsequently  mount  in  glycerin  gelatin.  Formalin  is  not 
to  be  used.  These  worms  are  most  likely  to  be  seen  as  writhing  thread- 
like worms,  especially  in  the  lymphatic  glands  and  connective  tissue, 
about  body  cavities. 

Fil'a'f  ift  medinensis.  —  The  Guinea  or  Medina  worm,  of  which 
until  recently  only  the  female  was  known,  is  of  great  importance  in 
parts  of  India,  Africa  and  Arabia.  The  female  is  a  thread-like  worm, 
about  20  to  30^  inches  long.  The  habitat  is  the  subcutaneous  and 


210 


THE    ROUND    WORMS. 


intermuscular  connective  tissue,  especially  of  the  lower  extremity. 
The  mouth  is  terminal  and  the  body  uniformly  cylindrical.  The 
uterus  is  a  continuous  tube  filled  with  sharp-tailed,  transversely 
striated  embryos,  650  x  17/4  and  constitutes  the  greater  part  of  the  body, 
the  alimentary  canal  being  pressed  to  one  side.  The  genital  organs 


13. 


/-OB. 


FIG.  65. — Round  worms  (Filariidae).  i,  Hooked  posterior  extremity  and  anterior 
extremity  of  Dracunculus  medinensis;  2,  cross  section  of  uterus  filled  with  embryos, 
D.  medinensis;  3  and  4,  free  embryo  and  embryos  of  D.  medinensis  in  intermediate 
host  (Cyclops);  5,  natural  size  of  female"  Filar'a  bancrofti;  6,  embryo  of  F.  ban- 
crofti  in  blood;  7,  tail  of  male  F.  bancrofti;  8,  male  and  female  of  F.  loa  (natural 
size);  9,  tuberculated  integument  and  posterior  end  of  male  F.  loa;  10,  posterior 
end  of  male  F.  perstans;  n,  male  of  F.  bancrofti  (natural  size);  12,  blunt  tailed 
embryo  of  F.  perstans;  13,  sharp  tailed  embryo  of  F.  demarquayi. 


probably  discharge  through  the  oesophagus.  The  body  when  being 
extracted  is  rather  transparent.  The  tip  of  the  tail  is  bent,  form- 
ing a  sort  of  anchoring  hook.  Recently  Leiper  fed  monkeys  on 
bananas  containing  infected  Cyclops,  and  at  the  autopsy  six  months 
later  obtained  both  male  and  female  forms. 


FILARIASIS.  211 

As  regards  the  life  history,  Fedschenko  showed  that  the  embryos 
when  liberated  swam  around  in  water  and  finally  entered  the  bodies  of 
species  of  the  genus  Cyclops.  The  female  tends  to  come  to  the  surface 
in  the  lower  extremities,  and  experiments  show  that  if  on  the  blister-like 
point  of  emergence  some  water  be  squeezed  out  from  a  sponge,  the 
uterus  will  eject  a  milky-looking  fluid  containing  myriads  of  embryos. 
This  would  indicate  that  the  worm  selects  the  lower  extremity  so  that 
the  embryos  may  gain  access  to  the  Cyclops  when  the  host  is  wading 
through  the  water. 

Leiper  showed  that  a  strength  of  HC1  equal  to  that  of  gastric  juice 
killed  the  Cyclops,  but  made  the  Dracunculus  embryos  very  active. 
From  this  he  judged  that  infection  must  probably  take  place  from 
drinking  water  containing  infected  Cyclops.  The  disease  is  known  as 
"Dracontiasis." 

Filaria  loa. — This  is  a  thread-like  worm,  about  i  to  2  inches  long. 
The  cuticle  is  characterized  by  distinct  wart-like  structures.  The 
males  are  smaller  than  the  females  and  have  3  preanal  papillae  and  2 
postanal  ones.  There  are  2  short  unequal  spicules.  The  life  history 
is  not  satisfactorily  established.  The  young  are  born  ovoviviparously, 
and  it  has  been  suggested  that  the  localized  cedemas,  known  as  Calabar 
swelling,  may  be  due  to  the  irritation  produced  by  these  eggs.  The 
embryos  almost  exactly  resemble  those  of  F.  bancrofti.  They  have  a 
diurnal  periodicity,  however,  appearing  in  the  blood  about  8  A.  M., 
increasing  to  noon  and  disappearing  about  9  P.  M.  The  intermediate 
host  is  unknown.  The  adult  worms  have  a  tendency  to  wrander  about 
in  the  subcutaneous  connective  tissue,  especially  about  the  region  of  the 
orbit  or  even  under  the  conjunctiva. 

Filaria  bancrofti. — This  is  the  most  important  of  the  filarial 
worms.  The  embryos  have  been  carried  in  medical  books  as  Filaria 
sanguinis  hominis.  This  species  is  the  cause  of  the  common  mani- 
festations of  filariasis,  such  as  elephantiasis,  varicose  groin  glands, 
chyluria,  lymph  scrotum,  etc. 

F.  bancrofti  is  transversely  striated,  and  lives  in  lymphatics  of  trunk 
and  extremities.  The  sexes  are  usually  found  together.  The  females 
are  about  3  inches  long  and  the  males  less  than  2  inches.  The  tails 
of  both  sexes  are  incurved,  but  that  of  the  male  is  more  so.  The 


212  THE    ROUND    WORMS. 

head  is  club-shaped.  The  sheathed  embryos  are  supposed  to  be 
born  viviparously  and  Manson  supposes  that  as  a  result  of  injury  to  the 
parent  worm  and  resulting  extrusion  of  eggs,  that  the  blocking  of  lymph 
channels  occurs.  These  embryos  show  a  nocturnal  periodicity.  Dur- 
ing the  day  they  remain  in  the  lungs.  The  disease  is  transmitted 
especially  by  Culex  fatigans.  The  sheathed  embryos,  getting  into 
stomach  of  mosquito,  wriggle  out  of  the  sheath,  they  then  bore  their 
way  through  walls  of  stomach  and  enter  into  a  sort  of  passive  stage, 
during  which  further  development  takes  place.  They  finally  become 
distributed  in  the  muscles  of  the  thorax  and  make  their  way  along  the 
fleshy  labium,  to  enter  the  wound  in  a  person  bitten  by  a  mosquito,  by 
way  of  Button's  membrane. 

Filaria  perstans. — The  adults  are  found  in  connective  tissue  and 
deeper  fat,  especially  about  the  mesentery  and  abdominal  aorta. 

The  female  is  about  3  inches  long;  the  male  is  rarely  found  and  is 
less  than  2  inches  long.  These  worms  are  characterized  by  incurved 
tails,  the  extremity  of  which  has  two  triangular  appendages  giving  a 
bifid  appearance.  The  embryos  do  not  possess  a  sheath  and  have  a 
blunt  tail.  The  life  history  is  unknown.  Both  mosquito  and  tick 
have  been  incriminated.  The  embryos  are  always  present  in  the 
peripheral  circulation — hence  perstans.  There  does  not  seem  to  be 
any  symptomatology. 

Filaria  demarquayi. — The  habitat  of  this  filarial  worm  is  the 
West  Indies.  The  embryo  has  no  sheath  and  has  a  sharp  tail.  Other 
filarial  species  which  have  been  reported  are  F.  magalhaesi,  F.  ozzardi, 
F.  volvulus,  F.  powelli  and  F.  philippinensis.  A  species  called  F.  gigas 
is  now  considered  to  have  been  only  the  hair  of  the  leg  of  a  fly.  The 
embryos  have  usually  been  given  such  names  as  F.  nocturna,  F.  diurna, 
etc.  Of  course  the  embryos  and  the  parent  should  have  the  same  name. 
It  has  been  proposed  to  designate  these  embryos  the  same  as  the  parent, 
but  with  the  use  of  the  term  Microfilaria  instead  of  Filaria. 

The  points  usually  noted  in  the  description  of  filarial  embryos  are : 

1.  Presence  or  absence  of  periodicity  of  embryos  in  peripheral 
circulation. 

2.  Presence  or  absence  of  a  sac  sheath  around  the  embryo. 


THE   WHIP   WORM.  213 

3.  Accurate  measurements. 

4.  Shape  and  description  of  head  and  tail  ends. 

5.  Character  of  movement. 

Key  to  Filarial  Larvae. 

A.  Sheath  present. 

1.  No  periodicity. 

F.  philippinensis.  Tightly-fitting  sheath;  not  flattened  out  beyond  extremi- 
ties. Tail  is  pointed  and  abruptly  attenuated.  Lashing  progression  move- 
ment. 320x6.5/4. 

2.  Periodicity  exhibited. 

a.  Nocturnal  periodicity. 

F.  bancrofti  (F.  nocturna).     Pointed  tail;  loose  sheath;  lashing  movement. 
300x7.5/4. 

b.  Diurnal  periodicity. 

F.  loa    (F.  diurna).  Pointed   tail;    loose   sheath;   cannot   be  distinguished 
from  F.  nocturna  except  by  periodicity.     300x7. 5/4. 

B.  Abscence  of  sheath.     None  of  these  exhibit  a  periodicity,  being  continuously 

present. 

1.  Blunt  tail — F.  perstans.     200x4.5/4. 

2.  Sharply-pointed  tail: 

a.  F.  demarquayi.     210x5/4. 

b.  F.  ozzardi.     215x5/4. 

NOTE. — A  filarial  embryo,  F.  powelli,  reported  once.  It  has  a  sheath,  nocturnal 
periodicity,  and  is  about  130x5/4. 

TRICHOTRACHELID^:. 

These  have  a  long  thin  neck  and  a  thicker  terminal  portion.  The 
oesophagus  is  of  the  single  row  of  cells  type.  The  anus  is  terminal; 
there  is  only  one  ovary. 

Trichocephalus  trichiurus. — This  is  usually  called  the  whip- 
worm — the  thickened  body  representing  the  handle  and  the  narrow 
neck  the  lash.  It  is  one  of  the  most  common  parasites  in  both  temper- 
ate and  tropical  climates.  The  egg  is  very  characteristic  in  having  an 
oval  shape  with  knobs  at  either  extremity.  It  resembles  a  platter  with 
handles.  The  male  is  almost  2  inches  long,  and  has  the  terminal 
portion  curled  up  in  a  spiral.  It  has  a  single  terminal  spicule. 

The  female  is  a  Jittle  longer  than  the  male,  and  has  the  terminal 
part  in  the  shape  of  a  comma  instead  of  being  coiled.  The  neck  only 
contains  the  cesophagus.  The  great  powers  of  resistance  of  the  ova 
may  account  for  their  general  distribution;  they  may  live  for  months 
under  conditions  of  freezing  and  so  forth.  There  is  no  intermediate 


214 


THE    ROUND    WORMS. 


host.  The  worm  arrives  at  sexual  maturity  in  about  one  month  after 
ingestion.  The  whip-worm  prefers  the  caecum,  but  also  lives  in  the 
lower  end  of  the  ileum  and  the  appendix. 

The  neck  burrows  into  the  mucosa,  and  much  importance  has  been 
attributed  by  the  French  to  the  possibility  of  this  paving  a  way  for  the 


FIG.  66. — Round  worms,  i,  Encysted  embryo  of  Trichinella  spiralis;  2,  male 
and  female  of  T.  spiralis;  3,  male  and  female  of  Trichocephalus  trichiurus;  4,  egg 
of  T.  trichiurus;  5  and  6.  head  and  male  and  female  of  Ascaris  canis;  7,  8  and  9, 
head,  egg  and  male  and  female  of  Oxyuris  vermicularis;  10,  n  and  12,  head,  egg 
and  tail  of  Ascaris  lumbricoides;  13  and  14,  head  and  egg  of  Echinorhynchusgigas; 
15,  1 6  and  17,  parthenogenetic  female  and  rhabditiform  and  filariform  embryos  of 
Strongyloides  stercoralis. 


entrance  of  pathogenic  bacteria.     They  do  not  seem  to  produce  serious 
symptoms. 

Trichinella  spiralis. — The  cause  of  trichinosis  is  usually  termed 
Trichina  spiralis  in  medical  works.  The  adults  live  in  the  duodenum 
and  jejunum;  the  males  are  about  1/20  of  an  inch  long  and  the  female 


TRICHINOSIS. 


215 


about  1/6  of  an  inch.  The  female  gives  off  embryos  from  the  vulva 
which  is  near  the  mouth  end  (viviparous).  After  fertilization  of  the 
females  the  males  die,  and  the  females  then  begin  to  produce  embryos 
to  the  number  of  more  than  1000  each.  These  pass  out  of  the  intestine 
and  wander  about  to  the  striated  muscle;  it  being  about  10  days  be- 
fore they  reach  the  muscle.  In  the  muscle  they  become  encysted  as 
the  oval  areas  containing  coiled-up  embryos  that  everyone  is  familiar 
with.  These  oval  areas  are  |about  450x250/4.  The  encysted  larvae 
may  remain  alive  as  long  as  10  to  20  years;  finally,  however,  the 
cyst  undergoes  calcareous  infiltration  and  the  embryo  dies.  Among 


FIG.  67. — Trichina  spiralis  (Zlegler). 

cannibals  it  would  be  easy  to  keep  the  cycle  going  by  eating  improperly 
cooked  or  raw  human  meat,  the  parasite  being  thus  transmitted. 

As  this  would  not  explain  the  transmission  among  civilized  men,  the 
following  is  the  life  history:  Man  obtains  his  infection  from  eating 
raw  pork,  the  embryos  encysted  in  the  muscle  of  the  hog  being  liberated 
in  the  stomach,  and  the  males  and  females  developing  in  the  intestine 
as  above  described.  The  hog  may  gain  his  infection  by  eating  the 
meat  of  other  hogs  or  rats.  These  rats  eat  scraps  of  pork  at  slaughter 
houses  and  become  infected.  In  man,  while  the  adults  are  breeding  in 
the  intestine,  we  have  gastrointestinal  symptoms.  About  10  to  20  days 
after  infection  the  embryos  begin  to  wander  and  we  have  the  acute 


2l6  THE    ROUND    WORMS. 

muscle  pains.  In  the  diagnosis  we  should  try  to  obtain  specimens  of 
the  pork  which  has  caused  the  trouble  in  order  to  examine  for  encysted 
Trichinae.  During  the  diarrhceal  stage  we  may  examine  the  stools  for 
adult  worms.  In  particular  examine  the  blood  for  eosinophilia. 

STRONG  YLID.E. 

In  this  family  the  male  has  a  caudal  bursa,  a  prehensile  sort  of 
expansion  at  the  posterior  end  for  copulatory  purposes. 

Eustrongylus  gigas. — This  is  the  largest  round  worm  infecting 
man;  it  is  usually  found  in  the  pelvis  of  the  kidney.  There  seem  to  be 
12  authentic  cases  of  infection  in  man.  The  females  are  about  40 
inches  long  and  about  1/3  of  an  inch  in  breadth.  The  copulatory 
bursa  of  the  male  distinguishes  it  from  Ascaris.  The  source  of  in- 
fection is  unknown.  The  very  characteristic  ova,  with  gouged-out 
oval  depressions,  may  be  found  in  the  urine. 

Trichostrongylus  instabilis. — This  is  a  small  strongyle  formerly 
known  as  Strongylus  subtilis.  The  male  is  about  1/6  of  an  inch  long, 
and  the  female  about  1/4  of  an  inch.  It  has  been  found  in  the  upper 
part  of  the  small  intestine  of  inhabitants  of  Egypt  and  Japan.  It  does 
not  appear  to  produce  symptoms. 

Agchylostoma  duodenale. — The  hook-worm,  so  called  for  the 
hook-like  projections  of  the  head  dorsally,  is  probably  the  most  impor- 
tant of  the  animal  parasites.  This  specimen  in  Europe  and  Africa  and 
the  Necator  americanus  in  the  new  world  cause  an  immense  amount  of 
invaliding.  The  Egyptian  anaemia  and  the  Porto  Rican  anaemia  are 
caused  by  this  parasite.  Hook-worms  may  be  found  in  the  small 
intestine  of  man  in  enormous  numbers.  They  either  produce  their 
effects  by  feeding  on  the  mucosa  or  by  causing  loss  of  blood.  The 
males  are  little  than  more  1/3  of  an  inch  long  and  the  females  little 
more  than  1/2  inch  in  length.  The  male  can  readily  be  distinguished 
by  his  umbrella-like  expansion  or  copulatory  bursa.  The  tail  of  the 
female  is  pointed.  The  mouth  of  the  Old  World  hook-worm  has  4  claw- 
like  teeth  on  the  ventral  side  of  the  buccal  cavity  and  2  on  the  dorsal 
aspect.  In  N.  americanus  the  ventral  teeth  are  replaced  by  chitinous 
plates.  The  copulatory  bursa  of  the  N.  americanus  is  also  different, 


HOOK   WORMS. 


217 


being  bipartite  in  the  division  of  dorsal  ray  rather  than  tripartite,  as 
with  the  A.  duodenale. 

The  delicate-shelled  eggs  pass  out  in  the  faeces  and  in  i  or  2  days  a 
rhabditiform  embryo  (200  x  14/4)  is  produced.  After  moulting  twice, 
it  remains  rather  quiescent;  it  is  at  this  stage  that  it  burrows  into  the 
skin  of  man,  producing  the  so-called  " ground  itch"  at  the  site  of 


7. 


FIG.  68. — Hookworm  anatomy,  i,  Rhabditiform  embryo  of  Strongyloides  ster- 
coralis  emerging  from  egg;  2  and  3,  egg  and  male  and  female  of  hookworm; 
4  and  5,  head  and  copulatory  bursa  of  male  Ancylostoma  duodenale;  6  and  7, 
head  and  copulatory  bursa  of  male  Necator  americanus.  Dorsal  ray,  N,  america- 
nus. shows  deep  cleavage  and  bipartite  tips. 


entrance.  Having  gained  access  to  the  lymphatics  and  veins,  they 
eventually  reach  the  lungs.  Here  they  get  into  the  bronchioles  and 
undergo  a  third  moulting.  They  then  wrork  their  way  up  the  trachea 
to  the  glottis  and  are  swallowed  to  then  become  adults  in  the  intestine. 
Dr.  Stiles,  \vhile  accepting  this  theory  of  the  life  history,  thinks  it 
15 


2l8  THE    ROUND    WORMS. 

probable  that  infection  is  also  brought  about  by  swallowing  directly  some 
infecting  stage. 

Necator  americanus.— This  is  the  species  of  hook-worm  found  in 
the  Southern  States  and  the  West  Indies.  It  is  very  prevalent  in  Guam, 
L.  I.  It  was  found  by  Looss  in  pigmies  from  Central  Africa,  so  that 
this  parasite  was  undoubtedly  brought  to  this  part  of  the  world  by 
slaves.  The  eggs  of  N.  americanus  are  larger  than  those  of  A.  duode- 
nale.  In  hook-worm  disease  we  have  ground  itch,  tibial  ulcer,  anaemia, 
interference  with  physical  and  mental  development  and,  in  bad  cases, 
dirt  eating. 

ASCARID^:. 

These  have  3  papillae  around  oral  cavity.  The  male  has  2  equal- 
length  spicules.  An  intermediary  host  is  not  needed  in  the  life  history 
of  this  family. 

Ascaris  lumbricoides. — The  male  worm  is  from  5  to  8  inches 
long  and  the  female  from  7  to  15  inches  in  length.  They  are  from  1/7 
to  1/4  of  an  inch  in  diameter.  The  body  of  the  worm  resembles  the 
ordinary  earth-worm,  but  is  more  grayish  than  red.  The  ova  are  very 
characteristic  with  a  rough  mammillated  exterior.  This  at  times  is 
shelled  off  and  we  have  a  smooth  egg  which  may  be  mistaken  for  eggs 
of  other  parasites.  The  eggs  leave  the  body  in  the  faeces  and  after  a 
long  time — a  few  weeks  to  several  months,  according  to  temperature- 
develop  an  embryo  which  remains  in  the  shell  until  swallowed  by  some 
man  or  animal.  It  is  stated  that  they  will  remain  alive  for  years.  On 
being  swallowed,  the  embryo  leaves  the  egg  and  we  have  males  and 
females  developing  in  the  intestine.  In  countries  where  such  parasites 
abound,  as  in  Guam  and  the  Philippines,  the  possibility  of  their  getting 
into  the  peritoneal  cavity  through  operative  measures  on  the  intestine 
must  always  be  thought  of. 

Ascaris  canis. — This  is  a  parasite  of  the  dog  and  cat,  but  is  oc- 
casionally found  in  children.  It  is  much  smaller  than  the  A.  lum- 
bricoides— male  is  2  to  3  inches  long,  female  4  to  5  inches  in  length. 
The  parasites  are  characterized  by  the  presence  of  wing-like  pro- 
jections from  the  anterior  end  (arrow-like  head). 

Oxyuris  vermicularis.— This  parasite  is  also  known  as  the  pin- 


THREAD   WORMS.  219 

worm,  seat-worm  or  thread-worm.  The  male  is  about  1/6  of  an  inch 
long  and  the  female  a  little  less  than  1/2  an  inch  in  length.  The  male 
has  a  single  spicule  and  the  female  a  long  tapering  tail.  The  eggs  are 
thin-shelled,  plano-convex  and  show  a  coiled-up  embryo.  After 
ingestion  of  eggs,  the  adults  develop  in  the  small  intestine  where  copu- 
lation takes  place;  the  males  then  die.  The  fertilized  females  go  to  the 
caecum  and  colon  where  they  remain  until  they  reach  maturity.  At 
this  time  the  females  wander  to  the  rectum  where  they  either  expel  their 
ova  or  themselves,  working  their  way  out  of  the  anus.  This  usually 
occurs  at  night,  and  the  scratching  induced  by  the  itching  causes  the 
eggs  to  be  widely  spread  about  the  region  of  the  anus.  The  worms 
may  also  wander  into  the  vagina,  urethra  or  under  prepuce.  It  will  be 
seen  that  as  a  result  of  the  scratching,  the  fingers  become  contaminated 
with  ova  which  may  be  carried  to  the  mouth  and  so  cause  a  fresh  in- 
fection. A  knowledge  of  the  life  history — the  early  location  in  the 
small  intestine,  and  later  on  in  the  large — shows  that  treatment  should 
be  dual  in  its  direction.  Enemata  for  the  gravid  female  in  the  rectum 
and  santonin  and  calomel  for  the  young  adults  in  the  small  intestine. 

ACANTHOCEPHALA. 

These  are  called  thorn-headed  worms  on  account  of  a  proboscis 
which  projects  like  a  little  peg.  It  has  several  rows  of  hooks  pro- 
jecting backward  which  enable  it  to  attach  itself  firmly  to  the  intestinal 
wall.  The  worm  absorbs  nourishment  through  the  general  body  wall, 
there  being  no  alimentary  canal  or  mouth.  These  worms  are  common 
in  hogs.  The  3  shelled  eggs  are  very  striking  and  the  intermediate 
stage  is  in  June  bugs. 

The  Echinorhynchus  or  Gigantorhynchus  gigas — E.  hominis 
E.  and  moniliformis — have  also  been  reported  for  man. 

HIRUDINEA  (LEECHES). 

Hirudino  medicinalis. — This  is  the  leech  used  medicinally  for  the 
abstraction  of  blood.  They  have  a  secretion  which  prevents  coagula- 
tion of  the  blood  so  that  when  removed  the  wound  still  continues  to 
bleed. 

Hirudo  nilotica. — This  species  has  been  found  in  many  parts  of 


22O  THE    ROUND    WORMS. 

Northern  Africa  and,  gaining  access  to  the  stomach  through  drinking 
water,  it  wanders  to  the  pharynx,  nares  and  even  trachea.  Manson 
refers,  to  a  case  of  obstinate  epistaxis  and  headache  caused  by  a  leech 
in  the  nostril. 

Haemadipsa  ceylonica. — These  are  land  leeches  found  in  India, 
Philippines,  Australia  and  South  America.  They  are  only  about  i 
inch  long  and  are  slender.  They  leave  the  damp  earth  to  climb  shrubs 
and  from  there  to  drop  on  animals  or  man  passing  through  the  forest. 
The  bites  are  painless,  but  may  be  followed  by  ulcers.  They  may  get 
into  the  nostrils. 


CHAPTER  XIX. 


THE  ARACHNOIDS. 


Order. 


Acarina, 


CLASSIFICATION  OF  THE  ARACHNOIDEA. 

Family.  Subfamily.  '     Genus.  Species. 

Trombidiidae,  Trombidium,          T.  holosericeum. 

Gamasidae,  Dermanyssus,          D.  gallinae. 

Tyroglyphidae,  TV™!™!,,,*         / T-  farinae> 

T.  longior. 


Sarcoptidae, 
Demodicidae, 


Ixodidae, 


Linguatulida, 


Sarcoptes, 
Demodex, 

Argas, 

Ornithodoros, 
Ixodes, 
Hyalomma, 
Rhipicephalus, 

Dermacentor, 

Margaropus, 
Amblyomma, 
Haemaphysalis, 
f  Linguatula, 
\  Porocephalus, 


THE  ARACHNOIDEA. 


Argasinae, 


Ixodinae, 


S.  scabiei. 

D.  folliculorum, 

A.  persicus. 

A.  miniatus. 

O.  saviguyi. 

I.  ricinus. 

H.  aegyptium, 

R.  bursa. 
f  D.  reticulatus. 
\  D.  andersoni. 

M.  annulatus. 

A.  hebraeum. 

H.  leachi. 

L.  rhinaria. 

P.  constrictus. 


The  Archnoidea  differ  from  the  Insecta  in  having  the  head  and 
thorax  fused  together.  They  also  have  4  pairs  of  ambulatory  ap- 
pendages, while  the  insects  only  have  3  pairs.  The  Arachnoidea  never 
have  compound  eyes — these  when  present  being  simple.  Of  the  2 
orders  of  Archnoidea  of  interest  medically  the  Acarina  is  far  more 
important  than  the  Linguatulida. 

ACARINA. 

Of  the  acarines  we  are  chiefly  interested  in  the  mites  and  the  ticks. 
The  acarines  do  not  show  any  separation  of  the  abdomen  from  the 
cephalo-thorax.  A  hexapod  larva  develops  from  the  egg;  this  is 
succeeded  by  an  octopod  nymph  which  differs  from  the  adult  in  not 
having  sexual  organs. 

221 


222  THE    ARACHNOIDS. 

The  Trombidiidae. 

These  generally  have  a  soft  integument  and  are  often  brightly  colored. 
A  very  common  and  annoying  member  of  this  family  is  the  hexapod  larva 
of  the  Trombidium  holosericeum.  It  is  usually  designated  Leptus 
autumnalis.  Popularly  it  is  termed  "harvest  mite,"  "red  bug"  or  jig- 
ger. They  are  found  in  the  fields  in  the  autumn  and  attack  both  man 
and  animals.  The  itching  and  redness  produced  is  at  times  called 
autumnal  erythema.  There  is  a  Trombidium  in  Mexico  which  has  a 
predilection  for  the  skin  of  the  eye-lids,  prepuce  and  navel.  The  Ked- 
ani  mite  is  believed  by  the  Japanese  authorities  to  bring  about  infection 
with  Japanese  river  fever  or  Tsutsugamushi,  as  the  result  of  trans- 
mitting either  a  bacterium  or  protozoon  by  its  bite.  The  disease  some- 
what resembles  typhus,  although  an  eschar  at  the  site  of  the  bite  and 
lymphatic  involvement  is  present.  Of  the  Gamasidae,  which  generally 
have  a  hard  body,  only  the  Dermanyssus  gallinae  is  of  interest.  This 
coleopterous  mite  infests  chicken-houses  and  sucks  the  blood  of  the 
inmates.  They  will  also  attack  man.  Poultrymen  may  be  troubled 
writh  a  sort  of  eczema  on  the  backs  of  the  hands  and  forearms,  similar 
to  scabies,  resulting  from  bites  by  these  mites.  They  measure  350  x  650^. 
They  have  no  eyes. 

Tyroglyphidae. 

Mites  of  this  family  live  on  cheese,  flour,  dried  fruits,  etc.  They  are 
chiefly  of  importance  because  of  their  being  occasionally  found  in 
urine,  faeces,  etc.,  and  being  striking  objects,  the  question  of  pathogen- 
icity  arises.  The  T.  longior  has  been  associated  with  intestinal  trouble 
(probably  a  coincidence,  patient  having  eaten  cheese  containing  these 
mites). 

Glyciphagi  are  found  in  sugar  and  are  the  cause  of  what  is  known 
as  "grocers'  itch."  Rhizoglyphus  parasiticus  is  reported  to  be  the 
cause  of  an  itch-like  affection  of  the  feet  of  coolies  on  tea  plantations. 
To  distinguish:  the  dorsum  of  Glyciphagus  is  hairy  or  plumose;  that 
of  Tyroglyphus  has  both  claws  and  suckers  on  tarsi,  while  Rhizoglyphus 
has  only  claws. 


ITCH   MITES. 


223 


Sarcoptidae. 

These  are  small  eyeless  mites  with  a  transversely  striated  cuticle. 
They  live  on  the  epidermis  of  man  and  various  animals.  It  is  the 
female  that  makes  the  tunnels  in  the  skin  between  the  ringers,  on  penis, 
flexor  surface  of  forearm,  etc.  The  male  dies  off  after  copulation. 
The  female  passes  through 4  stages:  (i)  larva;  (2)  nymph;  resembles 


7-oe, 


FIG.  69. — Mites,  i,  Demodex  folliculorum;  2,  Kedani  mite;  3,  Trombidium 
holosericeum;  4  and  6,  Female  and  male  Sarcoptes  scabiei;  5,  Tyroglyphus  farinae; 
7,  Rhizoglyphus  parasiticus. 

adult,  but  no  sexual  organs;  (3)  the  pubescent  female;  (4)  the  egg- 
bearing  female.  A  pair  of  itch  mites  may  produce  1,500,000  descend- 
ants in  3  months.  Transference  of  eggs,  larvae  or  pubescent  females 
do  not  seem  to  transmit  scabies.  It  is  the  egg-laden  female  only.  The 
human  itch  mite,  Sarcoptes  scabiei,  is  an  oval  mite,  the  male  is  250  x  150/4; 
the  female  is  about  400  x  300/4.  Besides-  the  difference  in  size,  the 
male  maybe  distinguished  from  the  female  by  the  fact  that  the  third  and 


224  THE    ARACHNOIDS. 

fourth  pairs  of  legs  in  the  female  have  bristles,  but  in  the  male,  the 
fourth  pair  has  suckers.  The  tunnels  made  by  the  female  have  the 
egg-bearing  female  at  the  blind  end;  scattered  all  along  are  faeces,  eggs, 
larvae;  the  eggs  being  next  the  mother  and  the  more  mature  young  at 
the  entrance  to  the  gallery.  A  diagnosis  can  be  made  from  the  finding 
of  either  eggs  or  larvae.  The  eggs  are  140/4  long  and  hatch  out  in  4  to 
5  days.  A  female  becomes  mature  in  about  3  weeks.  Different 
animals  have  different  species  of  itch  mites. 

Demodicidae  (Hair  follicle  mites). 

Demodex  folliculorum — This  is  a  vermiform  acarine  about  400/4 
long;  the  eggs  are  about  75/4  long;  they  chiefly  live  in  the  sebaceous 
glands  of  nose  and  forehead. 

Ixodidae. 

This  family  of  the  Arachnoidea  is  one  of  great  medical  interest  and 
of  growing  importance.  While  only  proven  the  intermediary  hosts  in 
the  case  of  the  organism  of  African  tick  fever  and  the  as  yet  undis- 
covered cause  of  spotted  fever  of  the  Rocky  Mountains,  there  is  con- 
siderable speculation  as  to  the  possibility  of  blackwater  fever  being  due 
to  a  Babesia  (piroplasma).  Piroplasmata  of  animals  seem  to  be 
invariably  transmitted  by  ticks. 

Very  important  diseases  due  to  these  small  pear-shaped  organisms 
within  red  cells  are  known  for  various  animals,  the  best  known  being 
that  of  cattle  in  Texas  and  known  as  Texas  fever.  Other  diseases  are 
Rhodesian  fever  (cattle),  heart  water  (sheep)  and  malignant  jaundice 
of  dogs.  In  these  diseases  there  are  pathological  features  which 
resemble  blackwater  fever  of  man.  It  is  of  interest  to  note  that  it  was 
with  the  transmission  of  Texas  fever  through  an  intermediate  host 
(the  tick)  that  Smith  and  Kilborne  (1889-1893)  established  the  zoologi- 
cal principle  of  transmission  of  disease  through  arthropod  interme- 
diary hosts.  This  led  up  to  the  work  on  malaria,  yellow  fever,  etc. 
Ticks  differ  from  insects  in  having  4  pairs  of  legs,  only  2  pairs  of 
mouth  parts  and  no  antennae.  They  differ  from  other  acarines  in 
having  a  median  probe-shaped  puncturing  organ,  the  hypostome, 
which  is  beset  with  numerous  teeth  projecting  backward.  The  head, 


TICKS.  225 

or  capitulum,  or  rostrum,  is  the  part  which  projects  anteriorly  from  the 
body.  This  carries  the  piercing  parts  which  are  the  single  hypostome 
and  a  pair  of  piercing  chitinous  structures,  the  chelicerae.  A  pair  of 
palpi  consisting  of  segments  are  on  either  side  of  the  biting  parts. 
Very  important  structures  are  the  stigmal  plates.  These  are  striking 
mosaic-like  areas  which  are  located  just  posterior  to  each  hind  leg  in 
the  Ixodinae  and  between  the  third  and  fourth  legs  in  the  Argasinae.  As 
the  greatest  confusion  exists  as  to  the  classification  of  ticks,  Dr.  Charles 
W.  Stiles  has  now  in  hand  a  system  of  classifying  ticks  according  to  the 
appearance  of  these  plates  as  seen  under  the  high  power  of  a  micro- 
scope. There  is  great  variation  in  the  outline  and  general  picture  of 
these  stigmal  plates  in  the  different  species.  The  stigmal  orifice,  the 
opening  of  the  tracheal  system,  is  in  the  center.  The  Ixodinae  have 
a  scutum  or  shield  like  chitinous  structure  on  the  dorsal  surface.  It 
covers  almost  the  entire  back  of  the  tick  in  the  male  and  only  a  small 
portion  in  the  female.  The  genital  opening  is  toward  the  anterior  part 
of  the  ventral  surface.  The  anus,  with  anterior  or  posterior  anal 
grooves,  is  near  the  posterior  third  of  the  venter. 

Life  History  of  Ticks. — This  varies  greatly  according  to  the  sub- 
family, genus  and  species.  The  female  Ornithodoros  savignyi  lays 
about  140  eggs.  The  larva  does  not  leave  the  egg,  but  moults  inside, 
and  finally  emerges  as  an  8-legged  nymph.  It  lives  in  the  dust  in  the 
cracks  of  the  native  huts  and  comes  out  at  night  to  feed  on  the  sleeping 
natives.  As  the  possibility  for  destruction  are  not  so  great  as  with 
many  Ixodinae  the  necessity  for  thousands  of  eggs  is  not  imperative  for 
the  continuation  of  the  species  as  with  the  Ixodinae.  With  some  of 
the  Ixcdinae  the  females  lay  from  5000  to  20,000  eggs  during  several 
days  or  weeks  and  then  die.  The  egg  is  preferably  deposited  near 
grass.  The  egg  stage  lasts  from  2  to  6  months,  when  the  6-legged 
larva  emerges.  Crawling  up  the  grass  a  passing  animal  is  gotten 
upon.  After  feeding,  the  larva  drops  to  the  ground,  and  changes  to  the 
pupal  stage  which  has  4  pairs  of  legs.  The  pupa  crawls  up  a  blade 
of  grass  and  gets  on  a  passing  animal  (the  second  one).  Feeding  it 
falls  to  the  ground  where  it  remains  8  to  10  weeks.  It  moults  and 
develops  into  an  adult  tick.  These  males  and  females  gain  access  to 
another  animal — the  males  fecundate  the  females,  after  which  the 


226 


THE    ARACHNOIDS. 


female  gorges  herself  with  blood;  afterward  dropping  off  the  animal 
and  laying  eggs.     With  some  ticks  fewer  hosts  suffice. 

Classification  of  Ixodidae. 

Subfamily  Argosinae. — Head  concealed  by  bcdy  when  viewed 
dorsally.  No  scutum.  Stigmal  plates  between  third  and  fourth  legs. 
Adults  have  no  suckers  beneath  claws. 


FIG.  70. — Ticks,  i,  Dorsal  aspect  of  female  Ixodinae;  2,  ventral  aspect  of  same; 
3,  dorsal  aspect  of  male  Ixodinas  showing  larger  scutum  or  shield;  4,  ventral  aspect 
of  same;  5  and  6,  ventral  and  dorsal  surface  of  Ornithodoros  moubata  (Argasinae); 
7,  mouth  parts  of  tick;  8,  Stigmal  plate  of  tick. 


Genus  Argas. — Body  narrow  in  front.  Margins  thin  and  acute. 
No  eyes.  The  A.  persicus  (Miana  bug)  of  Persia  has  been  supposed  to 
be  concerned  in  the  transmission  of  a  serious  disease. 

Genus  Ornithodoros. — Margins  of  bcdy  rounded.  Skin  has  many 
irregular  tubercles.  O.  savignyi  has  2  pairs  of  eyes  near  base  of 


TICKS.  227 

mouth  parts.  It  is  the  intermediate  host  of  Sp.  duttoni.  (South  African 
tick  fever.) 

Subfamily  Ixodinae. — Mouth  parts  project  in  front  of  body  when 
viewed  dorsally.  Scutum  present.  Stigmal  plates  posterior  to  fourth 
pair  of  legs.  Adults  have  suckers  on  claws. 

Section  Ixodce. — Transverse  recurved  preanal  groove  in  female. 
Genus  Ixodes. 

Section  Rhipicephalus . — No  preanal,  but  postanal  groove  in  female. 

In  the  genera  Aponomma  and  Hvalomma  the  palpi  are  long  and 
slender.  The  genus  Aponomma  has  eyes.  Amblyomma  has  no  eyes. 

Palpi  are  short  in  the  genera  Haemaphysalis,  Dermacentor  and 
Margaropus. 

Haemaphysalis  has  no  eyes;  Dermacentor  and  Margaropus  have 
eyes.  In  Dermacentor  the  head  is  transversely  oblong;  in  Margaropus 
it  is  hexagonal.  Dermacentor  andersonii  transmits  spotted  fever  of 
the  Rocky  Mountains. 

LlNGUATULIDA. 

These  are  vermiform  acarines  more  or  less  distinctly  annulated. 
They  have  hooks  at  either  side  of  the  mouth. 

Linguatula  rhinaria. — This  has  been  observed  in  man  both  in 
larval  and  adult  stages.  The  female  lays  eggs  which,  gaining  freedom 
through  the  nasal  mucus,  are  swallowed  by  various  animals.  A  larva 
develops  which  bores  its  way  through  the  gut  and  encysts  in  the  liver 
or  mesenteric  glands.  After  several  moultings,  they  work  their  way 
again  to  the  intestines  and  so  get  out  of  the  body  of  their  host;  or  they 
may  wander  to  lungs  and  trachea  and  either  escape  or  take  up  their 
position  in  the  nostrils  and  produce  eggs.  Consequently,  one  animal 
may  act  as  intermediate  and  definitive  host  or  these  cycles  may  take 
place  in  distinct  animal  hosts. 

Porocephalus  constrictus.— The  adult  form  lives  in  snakes  and 
the  eggs  are  probably  ingested  by  drinking  water.  These  eggs  de- 
velop into  a  curled-up,  ringed  larva,  which  is  encysted  especially  in  the 
liver  or  lungs.  These  escape  and  are  swallowed  by  the  snakes,  their 
definitive  hosts. 


CHAPTER  XX. 
THE  INSECTS. 

CLASSIFICATION  OF  THE  CLASS  INSECTA. 


Order.             Family.               Subfamily            Genus. 

Species. 

Rhynchota 
(Hemip- 
tera), 

Pediculidae, 
Acanthiidae, 

J  Pediculus, 

I  Phthirius, 
Acanthia, 

f  P.  capitis. 
\  P.  vestimenti. 
P.  pubis. 
A.  lectularia. 

Reduviidae, 

Conorrhinus, 

C.  sanguisuga. 

Pulicii 

iae'      f  Pnlex 

f  P.  irritans. 

j     JT  UlcXj 

|P.  cheopis. 

Pulicidae, 

]  Ceratophyllus, 
[  Ctneocephalus, 

C.  fasciatus. 
C.  serraticeps. 

Sarco] 

)syl-      Sarcopsylla, 

S.  penetrans. 

linae 

> 

Simulidae, 

Simulium, 

S.  damnosum. 

(buffalo  gnats), 

(mbwa). 

Psychodidse, 

Phlebotomus, 

P.  minutus. 

(sand  flies), 

Chironomidae, 

Ceratopogon, 

C.  pulicaris. 

(midges)              f  Culicinae,        Culex, 

C.  fatigans. 

Diptera, 

Culicidae,              -I  Anophe-         Anopheles, 

A.  maculipennis. 

[    linae, 

Tabanus,  ' 

T.  bovinus. 

Haematopota, 

H.  pluvialis. 

Pangonia, 

P.  beckeri. 

Tabanidae, 

Chrysops, 

C.  dispar. 

(horseflies) 

Glossina, 

G.  palpalis. 

Musca, 

M.  domestica. 

Auchmeromyia, 

A.  luteola. 

Muscidae, 

Calliphora, 

C.  vomitoria. 

Lucilia, 

L.  caesar. 

[  Chrysomia, 

C.  macellaria. 

(screw-  worm)  . 

Sarcophagidae, 

f  Sarcophaga, 
\  Ochromyia, 

S.  carnaria. 
O.  anthropophaga 

(Estridae, 

f  Dermatobia, 
\  Hypoderma, 

D.  cyaniventris. 
H.  diana. 

INSECTA. 

The  class  Insecta  belongs  to  the  branch  Arthropoda.     The  species 
belonging  to  it  far  outnumber  those  of  any  other  branch  of  the  animal 

228 


LICE.  229 

kindgom.  Besides  the  classes  Insecta  and  Arachnoidea,  we  have  the 
Crustacea  (crabs,  lobsters)  and  the  Myriapoda  (centipedes,  etc.).  The 
two  latter  are  of  very  little  importance  medically.  The  Arthropoda 
have  segmented  bodies,  but  they  differ  from  the  worms  in  having 
jointed  appendages  for  the  purposes  of  taking  in  food  and  moving  from 
place  to  place.  They  also  have  an  exoskeleton  which  is  more  or  less 
unyielding  from  the  deposit  of  chitin  in  the  cuticle. 

The  class  Insecta  have  one  pair  of  antennae,  3  pairs  of  mouth  parts 
(the  fused  labium  being  considered  as  one  pair),  and  3  pairs  of  legs. 
They  have  3  divisions  of  the  body — head,  thorax  and  abdomen.  The 
air  is  supplied  by  means  of  tracheae — branching  breathing  tubes. 
Insects  have  2  pairs  of  wings,  the  second  pair  of  which  is  frequently 
rudimentary  and  shows  simply  as  knob-like  projections.  These  are 
termed  halteres  or  balancers.  In  some  insects  both  pairs  of  wings  are 
rudimentary,  as  in  the  Aphaniptera.  Of  the  class  Insecta  only  the 
Rhynchota  (Hemiptera)  and  the  Diptera  are  of  special  importance. 

RHYNCHOTA. 

The  Rhynchota  are  sucking  insects  in  which  the  lower  lip  forms  a 
long  thin  tube  or  rostrum  which  can  be  bent  under  the  head  or  thorax. 
Inside  this  tube  are  biting  parts — mandibles  and  maxillae. 

The  Pediculidae. 

In  this  family  there  are  no  wings  and  there  is  no  metamorphosis. 
The  acorn-shaped  eggs  (nits)  are  deposited  on  hairs  of  the  host. 

Pediculus  capitis. — The  female  is  about  1/12  of  an  inch  long; 
the  male  smaller.  They  vary  in  color  according  to  the  color  of  the  hair 
of  the  host.  The  eggs  are  deposited  on  the  hairs  of  the  head.  The 
thorax  is  as  broa'd  as  the  abdomen.  There  seems  to  be  a  marked 
preference  exhibited  by  lice  for  their  own  peculiar  racial  host.  It  has 
recently  been  suggested  that  this  might  account  for  certain  peculiari- 
ties in  infection  where  different  races  were  living  together  and  under 
similar  conditions  as  to  food  and  environment,  and  yet  only  one  race 
contracts  the  disease.  (Beriberi.) 

Pediculus  vestimenti. — This  louse  lives  about  the  neck  and  trunk 


230 


THE    INSECTS. 


and  deposits  its  eggs  in  the  clothing.     It  is  almost  twice  the  size  of  the 
P.  capitis  and  the  abdominal  segment  is  broader  than  the  thorax. 

Phthirius  pubis. — This  louse  is  popularly  known  as  the  crab- 
louse.  The  female  is  little  more  than  1/25  of  an  inch  in  length,  and 
the  male  a  trifle  less.  They  are  almost  square.  They  have  a  pref- 
erence for  the  white  race  and  live  about  the  pubic  region.  The 
female  lays  about  a  dozen  eggs,  which  hatch  out  in  about  a  wreek. 


FIG.  71. — Important  Rhynchota.     i,  Head  louse;   2,  body  louse;  3   crab  louse; 
4,  bed  bug;  5,  mexican  bed  bug  (Conorhinus  sanguisuga). 

The  Acanthiidae. 

These  have  a  flattened  body,  a  3 -jointed  rostrum  and  4-jointed 
antennae.  Their  wings  are  atrophied. 

Acanthia  lectularia. — This  is  the  cosmopolitan  bed-bug.  It 
measures  about  1/5  by  i/ 8  of  an  inch.  It  is  of  a  brownish-red  color. 
The  bed^bug^lives  in  cracks  and  crevices,  especially  about  beds.  It  is 
said  they  can  migrate  from  house  to  house.  At  any  rate,  they  are 


BED-BUGS.  231 

frequently  transferred  with  wash  clothes.  They  have  a  penetrating 
odor.  The  eggs  are  deposited  in  cracks  and  in  10  days  they  hatch  out 
into  larvae  which  pass  insensibly  into  adults  by  a  series  of  moultings 
(5).  The  bed-bug  is  very  probably  the  intermediate  host  in  kala  azar, 
and  it  has  been  incriminated  in  connection  with  typhus  fever  and 
relapsing  fever. 

Reduviidae. 

Conorrhinus  sanguisuga. — This  is  known  as  the  Texas  or 
Mexican  bed  bug,  and  was  formerly  the  foe  of  the  common  bed-bug, 
but  having  gottten  a  taste  for  human  blood  through  the  Cimex  or 
Ancathia,  it  now  prefers  man.  It  is  extending  toward  the  North. 
It  has  wings.  The  bites  are  much  more  severe  than  those  of  the  com- 
mon bed  bug.  It  is  of  a  dark  brown  color,  nearly  an  inch  in  length, 
with  a  long,  flat,  narrow  head  and  a  short  thick  rostrum.  They  can 
run  as  well  as  fly.  They  bite  at  night. 

DIPTERA. 

The  order  Diptera  is  of  great  importance  medically  in  a  variety  of 
ways,  either  by  the  direct  irritation  of  their  bites,  by  their  transmitting 
disease  directly,  as  does  the  common  housefly  typhoid  fever,  or  by 
acting  as  intermediate  hosts  for  various  parasites.  They  are  character- 
ized by  mouth  parts  formed  for  puncturing,  sucking  or  licking.  They 
present  a  complete  metamorphosis,  larva,  pupa  and  imago.  As  a 
rule,  the  diptera  have  a  distinct  pair  of  wings,  the  second  pair  being 
rudimentary.  With  the  Aphaniptera  the  wings  are  practically  absent. 
Under  the  Aphaniptera,  \ve  have  to  consider  the  Pulicidae  or  flea 
family. 

Pulicidae. 

This  family  is  divided  into  2  subfamilies— the  Pulicinae  and  the 
Sarcopsyllinae.  In  the  former  the  female  remains  practically  un- 
changed after  fecundation,  in  the  latter  the  abdomen  becomes 
enormously  distended  with  eggs,  and  the  female  remains  stationary  after 
her  impregnation  in  the  burrow  which  she  has  made  under  the  skin. 


232  THE    INSECTS. 

Pulicinae. — Formerly,  with  the  exception  of  infection  with  D. 
canium,  the  fleas  were  only  under  suspicion  as  carriers  of  disease; 
ideas  having  been  entertained  as  to  their  being  possible  transmitters  of 
relapsing  fever,  typhus  fever  and  kala-azar.  As  a  result  of  the  con- 
vincing experiments  of  the  British  Plague  Commission,  their  role  in  the 
transmission  of  plague  has  been  absolutely  established.  It  is  by  the 
bite  of  the  Pulex  cheopis  that  plague  is  chiefly  transmitted  from  rat  to 
rat,  and  in  bubonic  and  septicaemic  plague  it  is  apparently  the  in- 
termediary in  human  infection.  The  puncturing  apparatus  of  the 
flea  consists  of  a  pointed  epipharynx  and  2  mandibles.  By  the  ap- 
position of  the  mandibles  to  the  epipharynx  a  tube  is  formed  through 
which  the  blood  is  sucked  up.  The  antennae  are  inconspicuous  and 
are  in  close  apposition  to  the  sides  of  the  head,  behind  the  eyes,  and  can 
only  be  well  made  out  with  a  lens.  Fleas  have  3  pairs  of  legs,  and  the 
male  can  be  distinguished  from  the  female  by  its  smaller  size  and  the 
conspicuous  coiled-up  penis  within  the  abdomen.  The  body  of  the 
flea  is  flattened  laterally.  They  may  or  may  not  have  eyes,  and  certain 
conspicuous  structures  called  combs  are  of  importance  in  classification. 
In  the  metamorphosis  of  the  flea  the  eggs  are  hatched  out  in  dust  of 
crevices,  etc.,  into  bristled  larvae  in  about  i  week.  The  larva  forms  a 
cocoon  and  develops  into"  a  nymph  which  has  3  pairs  of  legs.  The 
nymphs  emerge  from  the  cocoon  as  adult  fleas  in  about  3  weeks  after 
the  larva  forms  it. 

Key  to  the  Fleas. 

A.  No  eyes,  or  eyes  indistinct. 

1.  Densely  spinose.     Combs  on  some  abdominal  segments. 
Hystrichopsylla. 

2.  Combs  on  head  and  prothorax,  none  on  abdomen.  Typh- 
lopsylla. 

B.  Eyes  present. 

1.  Combs  along  inferior  border  of  head  and  posterior  border 
of  prothorax.     Ctenocephalus. 

2.  No  combs  along  inferior  border  of  head,  but  on  posterior 
border  of  prothorax.     Ceratophyllus. 

3.  No  combs.     Pulex. 


FLfcAS. 


233 


The  common  house  flea  of  Europe  is  the  Pulex  irritans;  that  of  the 
Tinted  States  the  Ctenocephalus  serraticeps  or  dog  flea.  The  flea  that 
is  implicated  with  plague  is  the  Pulex  cheopis.  It  resembles  P.  irritans, 
but  is  more  yellow  than  brown  in  color.  It  also  has  a  greater  number 
of  bristles  on  the  head.  The  ocular  bristle  runs  above  and  in  front  of 


FIG.  72. — Various  pulicidae.  i,  Ceratophyllus  fasciatus  (rat  flea);  2,  Pulex- 
cheopis  (plague  transmitting  flea);  3,  Ctenocephalus  serraticeps  (dog  flea);  4,  Sar- 
copsylla  penetrans  (chigoe);  5,  head  of  flea  showing  palps;  6,  female  of  S.  pene- 
trans  distended  with  eggs  after  burrowing  under  skin. 


the  eye;  that  of  P.  irritans  below.  It  is  principally  the  flea  of  the  M. 
decumanus,  the  sewer  rat;  but  the  house  rat,  M.  rattus,  becomes  in- 
fected from  coming  in  contact  \vith  the  sewer  rat  in  the  basement. 

Sarcopsyllinae. 

Belonging    to     the    subfamily    Sarcopsyllinae,    the    Sarcopsylla 
penetrans  is  of  great  importance  in  tropical  countries.     It  is  known  as 
16 


234  THE    INSECTS 

the  chigoe,  nigua  or  jigger.  The  male  is  unimportant.  The  female, 
which  when  unimpregnated  is  only  about  1/24  of  an  inch  long,  when 
impregnated  bores  its  way  into  the  skin  of  man,  especially  about  the 
toes,  soles  of  the  feet  or  finger  nails,  and  in  the  chosen  site  develops 
enormously,  becoming  as  large  as  a  small  pea.  A  small  black  spot  in 
the  center  of  a  tense  rather  pale  area  is  characteristic.  The  abdomen  of 
the  female  is  filled  with  eggs.  The  metamorphosis  is  similar  to  that  of 
the  flea.  Sarcopsylla  can  be  differentiated  from  the  flea  by  the  pro- 
portionately larger  head  to  the  body,  and  especially  by  the  fact  that  the 
head  is  the  shape  of  the  head  of  a  fish,  distinctly  pointed.  With  the 
fleas  the  lower  border  of  the  head  comes  out  in  a  straight  line  to  join 
the  curve  of  the  upper  part.  In  the  Sarcopsylla  lower  and  upper 
border  of  head  are  both  curved. 

Tabanidae. 

This  is  the  family  of  horseflies  or  gadflies.  It  is  the  most  numer 
ous  family  of  the  Diptera — there  being  more  than  1000  species. 
The  females  are  blood  suckers;  the  males  live  on  flowers.  They  are 
large  and  stockily  built.  The  eyes  are  usually  very  brilliant  in  color, 
and  in  the  male  make  up  the  greater  part  of  the  head.  The  eggs  are 
laid  on  leaves;  the  larva  is  carniverous;  the  pupa  free. 

Tabanus  autumnalis. — Is  about  3/4  of  an  inch  long;  it  is  dark  in 
color,  and  has  4  longitudinal  bands  on  the  thorax.  •  The  last  joint  of 
the  antennae  has  a  crescentic  notch.  The  wings  do  not  overlap.  No 
spurs  at  tip  of  hind  tibia. 

Haematopota  pluvialis.— In  the  Haematopota  there  is  no 
crescentic  antennal  notch,  and  the  wings  overlap.  The  abdomen  is 
narrower  than  in  Tabanus.  They  also  have  spurs  on  hind  tibia. 
The  brimp,  one  of  the  Haematopota,  bites  man  severely. 

Pangonia  beckeri. — The  genus  Pangonia  is  characterized  by  a 
very  long,  slender  and  more  or  less  horizontal  proboscis. 

Chrysops  dispar. — Chrysops  and  Pangonia  have  spurs  at  tip  of 
hind  tibiae.  The  wings  are  widely  separated  and  spotted.  The 
antennae  of  Chrysops  are  especially  long  and  slender.  Chrysops  and 
Haematopota  produce  the  greatest  amount  of  pain  from  their  bites. 


BITING    FLIES. 


235 


The  Tabanidae  are  not  implicated  as  intermediate  hosts  in  the  trans- 
mission of  disease.  By  their  bites,  however,  they  may  transmit  disease 
directly,  as  with  anthrax. 

Muscidae. 

The  common  housefly,  M.  domestica,  is  the  best  example  of  this 
family.     This  fly  is  incapable  of  biting,  but  may  transmit  disease 


\ 


7- oft. 


FIG.  73. — Insects  in  which  imago  stage  is  important,  i,  Stomoxys  calcitrans; 
2,  arista  of  Glossina  palpalis;  3,  Glossina  palpalis  (tsetse  fly).  4,  Tabanus  autum 
nalis. 


directly,  carrying  infectious  material  from  the  source,  as  in  faeces,  to  the 
food  about  to  be  ingested.  Their  role  in  typhoid  fever  is  one  of  im- 
mense importance.  • 

In  the  Muscidae  the  antennae  hang  down  in  front  of  the  head  in  3 
segments,  and  have  a  plumose  arista.  There  are  no  bristles  on  ab- 
domen except  at  tip. 


236  THE    INSECTS. 

Stomoxys  calcitrans. — These  greatly  resemble  the  common 
housefly  in  size  and  shape.  They  can  be  easily  distinguished  by  the 
black,  piercing  proboscis  extending  beyond  the  head.  There  are 
longitudinal  stripes  on  the  thorax  and  spots  on  the  abdomen.  The 
proboscis  on  examination  will  be  seen  to  be  bent  at  an  angle  near  its 
base.  The  palps  are  short  and  slender.  The  wings  diverge  widely. 
The  genus  Stomoxys  includes  vicious  biters.  This  is  the  fly  which 
comes  into  houses  before  a  rain,  and  which  has  given  the  common 
housefly  the  reputation  of  biting  before  a  rain.  Stomoxys  may  be 
implicated  in  transmitting  surra  (Trypanosoma  evansi). 

The  horsefly  (Haematobia  irritans)  rarely  bites  man.  In  these  the 
palpi  are  much  longer  than  in  Stomoxys. 

Glossina  palpalis. — This  is  the  tsetse  fly  that  is  responsible  for 
the  transmission  of  human  trypanosomiasis  and  the  later  stage  of  the 
disease,  sleeping  sickness.  The  tsetse  fly  is  a  small  brownish  fly  about  1/3 
of  an  inch  long.  The  proboscis  extends  vertically  and  has  a  bulb  at  its 
base.  The  arista  is  plumose  only  on  the  upper  side.  The  wings  are 
carried  flat,  closed  over  one  another,  like  scissor  blades.  The  most 
characteristic  feature  of  the  tsetse  fly  is  the  way  the  fourth  longitudinal 
vein  bends  up  abruptly  to  meet  the  oblique  transverse  vein.  In 
Stomoxys,  the  wings  separate;  in  Hsematopota  they  just  meet,  and  in 
Glossina  they  cross.  The  tsetse  fly  does  not  lay  eggs,  but  gives  birth  to 
a  single  full-grown  larva  which  immediately  becomes  a  pupa.  Glos- 
sina morsitans  transmits  the  cattle  trypanosomiasis  disease,  nagana. 

Auchmeromyia  luteola. — This  is  an  African  fly,  the  larva  of 
which  is  known  as  the  "  Congo  floor  maggot,"  and  is  a  blood  sucker. 
The  larva  is  of  a  dirty -white  color  and  about  2/3  of  an  inch  long.  It 
crawls  out  at  night  and  feeds  on  the  sleeping  natives.  This  is  the  only 
known  instance  of  a  blood-sucking  larva. 

Calliphora  vomitoria  and  Lucilia  caesar. — These  are  flies  with 
brilliant  metallic-colored  abdomens,  commonly  called  blue-bottle  flies. 
They  deposit  their  eggs  on  tainted  meat  and  in  wounds.  Many  cases 
of  obscure  abdominal  trouble  are  probably  due  to  the  larvae  of  these 
flies.  Intestinal  myiasis  is  undoubtedly  of  greater  importance  than  has 
been  thought.  The  larvae,  with  hook-like  projections  anteriorly  and  a 
ringed  body,  can  easily  be  recognized  in  the  faeces.  They  have  been 


MYIASIS. 


237 


mistaken  for  flukes.     They  also  have  a  tendency  to  be  attracted  by 
those  with  ozena  and  the  larvae  may  develop  in  the  nostrils. 

Chrysomyiamacellaria. — This  is  known  as  the  screw-worm  when 
in  the  larval  stage.  The  adult  fly  resembles  the  blue-bottle  flies.  The 
thorax  is  striped.  The  eggs,  which  number  250  or  more,  when 
deposited  in  the  nostrils  or  in  wounds,  develop  into  the  screw-worm 
larvae,  which  may,  by  going  up  into  the  frontal  sinus,  cause  death. 


FIG.  74. — Insects  in  which  larval  stage  is  important,  i  and  2,  Insect  and  larval 
forms  of  Dermatobia  cyaniventris;  A,  ver  macaque,  B,  Torcel  or  Berne;  4  and  6, 
Insect  and  larva  of  Calliphora  vomitoria;  5  and  7,  insect  and  larva  of  Compso- 
myia  macellaria  (screw  worm). 


Sarcophagidae. 

These  are  known  as  "flesh  flies." 

Sarcophaga  carnaria. — This  is  a  grayish  fly  with  3  stripes  on 
thorax  and  black  spots  on  each  segment  of  the  abdomen.  It  is  vivi- 
parous. The  larvae  gain  access  to  nasal  and  other  cavities  and  there 


238  THE    INSECTS. 

develop.  Cases  of  death  have  been  reported.  Naturally,  the  fly 
deposits  its  larvae  on  decaying  flesh.  In  times  of  war  all  of  these  flies 
become  important  by  reason  of  "maggots"  in  the  wound. 

Ochromyia  anthropophaga. — This  is  an  African  fly  whose  larvae 
develop  under  the  skin  of  man  and  animals.  It  is  known  as  the  Ver 
de  Cayor.  The  larva  resembles  the  Ver  Macaque. 

(Estridae. 

Dermatobia  cyaniventris. — These  are  large,  thick-set  flies,  with 
prominent  head  and  eyes,  small  antennae  and  a  marked  narrowing  at 
the  junction  of  thorax  and  abdomen.  The  abdomen  is  a  metallic  blue. 
The  larvae  are  deposited  under  the  skin  in  various  parts  of  the  body. 
When  the  larvae  move  they  cause  considerable  pain.  At  first  the  larva 
is  club-shaped,  but  later  on  it  becomes  oval.  The  former  is  called  Ver 
Macaque,  the  latter  Torcel. 

Hypoderma  diana. — The  larval  form  of  this  fly  has  been  reported 
3  times  for  man. 


CHAPTER  XXI. 
THE  MOSQUITOES. 

MOSQUITOES  (Culicidae)  are  of  the  greatest  importance  medically, 
not  only  from  their  influence  upon  health  in  general  by  reason  of  inter- 
ference with  sleep  and  possibly  from  direct  transmission  of  disease,  but, 
more  specifically,  they  are  the  only  means  by  which  it  at  present  appears 
possible  to  bring  about  infection  with  such  diseases  as  yellow  fever, 
malaria,  filariasis  and  possibly  dengue.  In  addition,  many  diseases 
of  animals  are  transmitted  by  mosquitoes. 

The  Culicidae  differ  from  all  other  Diptera  in  having  scales  on  their 
wings  and  generally  on  head,  thorax  or  abdomen. 

To  identify  a  mosquito,  examine  a  wing  and  note  the  scales;  also 
note  the  presence  of  two  distinct  fork  cells  and,  in  addition,  that  the 
costal  vein  passes  completely  around  the  border  of  the  wing,  making  a 
sort  of  fringe  with  its  scales.  Mosquitoes  undergo  a  complete 
metamorphosis,  there  developing  from  the  egg  a  voracious,  rapidly- 
growing  larva;  next,  a  nongrowing,  nonf ceding  stage — the  pupa  or 
nymph.  There  the  head  and  thorax  are  combined  in  an  oval  body, 
from  the  back  of  which  projects  the  syphon  tubes;  and,  tucked  in 
ventrally  is  a  small  tail-like  appendage. 

The  fully  developed  insect  emerges  from  the  pupa. 

The  principal  mosquito -like,  blood-sucking  Diptera  which  are 
frequently  mistaken  for  mosquitoes — none  of  which  have  scales  on  their 
wings — are  the  following: 

i.  Chironomidae  or  Midges. — The  blood-sucking  species  of 
Chironomidae,  which  are  found  in  most  parts  of  the  wrorld, 
belong  chiefly  to  the  genus  "Ceratopogon."  These 
midges  are  of  very  small  size,  about  1/12  of  an  inch  long, 
are  able  to  get  through  netting  and,  usually  being  in 
swarms,  they  are  exceedingly  troublesome.  One  of  the 

239 


240  THE    MOSQUITOES. 

midges,  the  "jejen"  of  Cuba,  is  a  great  scourge,  its  small 
size  enabling  it  to  enter  eyes  and  nostrils.  The  larva  of 
Chironomus  is  a  red  worm -like  creature;  the  pupa  has  a 
tufted  head. 

2.  Simulidae    or    Buffalo    Gnats. — These  are  small  blood- 
thirsty insects  only  about  1/8  of  an  inch  in  length.     The 
thorax  is   humped,  the  legs  are  short  and  the  proboscis 
short  and  inconspicuous. 

One  species,  the  S.  damnosum,  known  by  the  natives  of 
Uganda  as  "Mbwa,"  is  greatly  dreaded;  its  bites  causing 
swellings  and  sores. 

3.  Psychodidae. — These  are  small,  hairy,  slender  midges, 
with  long   legs   and   a   long   proboscis.     They   are   only 
about  if 1 2  of  an  inch  in  length. 

Mosquitoes  have  3  main  parts  of  the  body — the  head,  the  thorax  and 
the  abdomen.  On  the  head,  the  space  behind  the  two  compound  eyes 
is  called  the  frons,  in  front,  and  the  occiput  posteriorly. 

The  nape  is  back  of  the  occiput.  The  bulbous  prolongation  of  the 
frons  which  projects  over  the  attachment  of  the  proboscis  is. the 
clypeus.  The  clypeus  is  hairy  in  the  Culex;  scaly  in  Stegomyia.  The 
proboscis  is  straight  in  all  mosquitoes  of  importance  medically.  It 
consists  of  a  fleshy,  scaled,  gutter-shaped  portion  beneath,  known  as  the 
labium,  which  terminates  in  two  hinge-joint  processes — the  labella. 
At  the  end  of  the  labium  is  a  thin  membrane  (Button's  membrane). 
It  is  through  this  that  filarial  embryos  are  supposed  to  pass  on  their  way 
from  the  interior  of  the  labium  to  enter  the  person  bitten.  The  labium 
may  be  considered  as  the  sheath  of  a  knife,  holding  and  protecting  the 
slender,  blade-like  penetrating  organs.  Lying  in  this  groove  we  have, 
from  above  downward,  the  horseshoe-shaped  labrum — epipharynx, 
the  under  surface  of  which  is  open.  This  when  closed  by  the  under- 
lying hypopharynx  forms  a  tube  through  which  the  blood  is  sucked  up 
by  the  mosquito.  In  the  hypopharynx,  which  somewhat  resembles  a 
hypodermic  needle,  is  a  channel,  the  veneno-salivary  duct.  It  is  down 
this  channel  that  the  malarial  sporozoite  passes.  There  are  2  pairs  of 
mandibles  and  2  pairs  of  maxillae  on  either  side  of  the  hypopharynx — 


MOSQUITOES. 


241 


the  mandibles  above  and  the  maxillae  below.  The  serrations  of  the 
maxillae  are  coarser  than  those  of  the  mandibles.  The  sensory  organs, 
the  palps,  lie  on  either  side  of  and  slightly  above  the  proboscis.  These 
are  of  the  utmost  importance  in  differentiating  mosquitoes  and  must  not 
be  confused  with  the  antennae,  which  are  attached  above  the  palpi  and 
at  the  sides  of  the  clypeus.  These  antennae  are  of  importance  in  dis- 
tinguishing the  sex  of  the  mosquito. 


FIG.  75. — Anatomy  of  mosquito,  i,  Dorsal  view  of  mosquito;  2,  wing  of 
mosquito;  A,  costal  vein;  B,  mid  cross  vein;  C,  posterior  cross  vein;  D,  first  fork- 
cell;  E,  second  fork-cell;  3,  various  types  of  scales;  a,  flat  head  scales;  b  and  c, 
Mansonia  wing  scales;  d,  upright  forked  head  scales;  e  f ,  g  and  h,  various  shapes 
of  thoracic  scales. 


The  thorax  is  largely  made  up  of  the  mesothorax,  at  the  posterior 
margin  of  which  is  a  small,  sharply -defined  piece,  the  scutellum;  this 
may  be  smooth  or  trilobed.  Underneath  and  posterior  to  the  scutellum 
is  the  metanotum;  the  metanotum  is  bare  in  Culicinae,  has  hairs  in 
Dendromyinae  and  scales  in  Joblotinae. 


242  THE    MOSQUITOES. 

There  is  a  pair  of  wings  attached  to  the  posterior  part  of  the 
mesothorax  and,  more  posteriorly  still,  a  pair  of  rudimentary  wings 
(halteres)  attached  to  the  metanotum.  The  3  pairs  of  legs  are  at- 
tached to  the  thorax. 

There  are  9  segments  in  the  abdomen.  The  genitalia  arise  from 
the  terminal  segment  as  bilobed  processes.  In  the  male  there  is  a  pair 
of  hook-like  appendages  or  claspers,  between  which,  and  ventrally 
situated,  are  the  harpes,  also  a  pair  of  chitinous  processes. 

In  considering  the  question  of  the  possible  danger  which  might 
arise  from  the  introduction  of  a  case  of  yellow  fever,  malaria  or  filariasis, 
it  would  give  the  greatest  information  if  the  ova  were  at  hand  so  that  we 
could  by  watching  the  development  from  egg  to  larva,  pupa  and  insect, 
have  all  the  points  from  which  to  decide  as  to  the  genera  developing  in 
the  given  locality.  It  is  generally  a  very  easy  matter  to  dip  out  large 
numbers  of  larvae  from  the  pools  and  having  noted  the  characteristics 
of  the  larvae,  to  do  the  same  when  the  pupae  develop;  so  that  we  have 
only  to  verify  our  identification  when  the  insect  emerges  from  the  pupa. 

THE  OVA. 

The  egg  raft  of  Culex,  containing  about  250  ova,  is  quite  perceptible 
on  the  surface  of  the  water  as  a  black,  scooped-out  mass,  about  1/5  of  an 
inch  in  length.  The  eggs  are  set  vertically  in  the  raft.  The  eggs  of 
the  Stegomyia  are  laid  singly  and  have  a  pearl-necklace-like  fringe 
around  them. 

The  Anophelinae  eggs  are  oval  in  shape  with  air-cell  projections 
from  either  side.  They  are  laid  in  triangle  and  ribbon  patterns.  The 
markings  of  these  air  cells  vary  and  have  been  used  for  differentiation. 
The  length  of  time  of  the  egg  stage  varies  according  to  temperature 
and  other  conditions — i  to  3  days  for  Stegomyia  and  2  to  4  days  for 
Anophelinae.  The  Anophelinae  are  more  difficult  to  raise  than 
Culex  or  Stegomyia. 

LARV.E. 

There  are  two  great  classes  of  larvae — the  siphonate  and  the 
asiphonate.  The  latter  are  always  Anophelinae. 

The   Culicinae    larvae    have  a    projecting  breathing  tube  at  the 


MOSQUITOES. 


243 


posterior  extremity  which  is  called  a  respiratory  syphon.  This  pro- 
jects off  at  an  angle  from  the  axis  of  the  body,  the  true  end  of  which 
terminates  in  four  flap-like  paddles.  If  you  divide  the  length  of  the 
syphon  by  the  breadth,  you  get  what  is  known  as  the  syphon  index. 
In  Culex  the  syphon  is  long  and  slender;  in  Stegomyia  it  is  short  and 
barrel-shaped.  When  at  the  surface  the  Culex  larva  has  his  syphon 
almost  vertical  and  the  body  at  an  angle  of  about  45°. 


A-H.Ebfclinpf  ?-«>« 


FIG.  76. — Metamorphosis  of  mosquitoes,  i,  2,  3,  4  and  5,  Eggs,  larva,  pupa 
and  heads  of  male  and  female  Culex;  6,  7,  8,  9  and  10,  eggs,  larva,  pupa  and  heads 
of  male  and  female  Anopheles;  n,  12,  13  14  and  15.  Eggs,  larva,  pupa  and  heads 
of  male  and  female  Stegomyia. 

The  Stegomyia  larva  hangs  more  vertically.  As  a  rule,  the  hairs 
proceeding  from  the  sides  of  Culex  larvae  are  straight  and  the  head 
relatively  large.  There  are  also  no  palmate  hairs  along  the  sides. 

The  Anophelinae  larvae  have  a  small  head  which  is  capable  of 
being  twisted  around  with  lightning-like  rapidity.  They  are  darker  in 
color  and  have  no  syphon;  float  parallel  to  the  surface  of  the  water; 


244  THE    MOSQUITOES. 

have  long  lateral  branching  hairs,  and  on  the  sides  of  each  of  the  5  or  6 
middle  abdominal  segments  they  have  a  pair  of  palmate  hairs.  These 
palmate  hairs  are  supposed  to  aid  them  in  keeping  their  position  on  the 
surface  of  the  water.  The  larvae  are  usually  called  "wigglers." 
The  duration  of  the  larval  stage  is  from  i  to  2  weeks,  according  to  the 
temperature. 

THE  PUP^:. 

These  have  a  bloated-looking  cephalo-thorax  and  a  shrimp-like 
tail — the  latter  the  abdomen.  Very  important  in  examining  them  with 
a  lens  is  to  note  the  characteristics  of  the  syphon  tubes  which  project 
from  the  dorsal  surface.  These  syphons  are  long  and  slender  in 
Culex  and  project  from  the  posterior  portion  of  the  head  end.  In 
Anophelinae  they  are  broadly  funnel-shaped  and  arise  from  the 
middle  of  the  head  end.  The  syphon  of  the  Stegomyia  is  triangular. 

The  bulbous  end  of  the  Culex  nymph  is  more  vertical  than  the 
horizontally-placed  cephalo-thorax  of  Anopheles.  The  duration  of 
pupal  life  is  short — only  1-3  days.  At  the  end  of  this  time  the  pupa 
comes  to  the  surface  and  straightens  out.  The  integument  then 
splits  dorsally  and  the  perfect  insect  emerges.  It  dries  its  wings  for  a 
time  on  its  raft-like  pupal  skin  and  then  flies  away. 

From  the  above  it  will  be  seen  that  the  stages  in  the  metamorphosis 
of  the  mosquito  takes  about  2  weeks:  1-3  days  for  egg  stage;  7-10  days 
for  larval  stage  and  2-3  days  for  pupal  stage. 

DISSECTION  OF  THE  MOSQUITO. 

The  easiest  way  to  secure  a  mosquito  for  dissection  is  to  use  an 
ordinary  plugged  test-tube.  Slipping  the  open  end  of  the  test-tube  over 
the  resting  mosquito;  by  a  slight  movement,  the  insect  will  fly  toward 
the  bottom.  Then  quickly  insert  the  plug.  If  it  is  not  desired  to 
study  the  scales,  the  best  way  to  kill  the  mosquito  is  by  striking  the  tube 
sharply  against  the  thigh.  If  it  is  also  desired  to  study  the  scale 
characteristics  it  is  better  to  put  a  drop  or  so  of  chloroform  on  the  lower 
part  of  the  cotton  plug.  The  vapor  falls  to  the  bottom  of  the  tube  and 
kills  the  mosquito.  Take  the  mosquito  out,  pull  off  legs  and  wings  and 


MOSQUITOES. 


245 


then  place  the  body  in  a  drop  of  salt  solution  on  a  slide.  Then  hold 
the  anterior  end  of  the  thorax  by  pressure  of  a  needle.  With  a  needle 
in  the  other  hand,  gently  crush  the  chitinous  connection  between  the 
sixth  and  seventh  segments  of  the  abdomen.  Then  holding  the  thorax 
firm,  steadily  and  gently  pull  the  last  segments  in  the  opposite  direction. 
If  this  is  done  properly,  a  delicate  gelatinous  white  mass  will  slowly 
float  out  in  the  salt  solution.  One  should  be  able  to  secure  the  aliment- 
ary canal  as  far  up  as  the  proventriculus,  which  is  just  anterior  to  the 


FIG.  77. — Anatomy  of  mosquito,  i,  Cross  section  of  proboscis  of  mosquito; 
2.  anatomy  of  mosquito  longitudinal  section;  3,  tip  of  proboscis  of  mosquito; 
a,  labrum-epipharynx;  b,  hypopharynx;  c,  mandible;  d,  maxilla. 

stomach,  the  part  in  which  the  malarial  zygotes  develop.  Proceeding 
from  before  backward,  we  have  the  proventriculus,  which  is  a  sort  of 
muscular  ring  at  the  opening  of  the  stomach  or  mid-gut.  Leading 
from  the  stomach  we  have  the  hind  gut,  which  ends  in  the  rectum. 
Taking  origin  at  the  posterior  end  of  the  stomach  and  festooning  the 
hind  gut  are  5  longitudinal  tubes— the  Malpighian  tubules.  These  are 
characterized  by  large  granular-like  cells  with  a  prominent  refractile 
nucleus.  They  are  regarded  as  the  renal  structures.  It  is  in  these 
tubules  that  the  embryo  of  the  Filaria  immitis  of  the  dog  develops. 


246  THE    MOSQUITOES. 

In  the  female  mosquito,  the  parts  withdrawn  may  seem  to  be  largely 
made  up  of  the  white  oval  ovaries.  These  are  connected  with  the 
spermathecae,  in  which  the  spermatozoa  are  stored  after  fecundation  by 
the  male.  In  the  male  the  testicles  are  quite  distinct.  Next  to  the 
examination  of  the  stomach  for  zygotes,  which  appear  as  wart-like 
excrescences  on  its  outer  surface,  the  most  important  structures  are  the 
salivary  glands,  where  the  malarial  sporozoites  are  found.  The 
easiest  way  to  dissect  out  the  salivary  glands  is  to  press  down  firmly,  but 
gently,  on  the  anterior  part  of  the  thorax,  and  then  with  the  shaft  of  a 
second  needle,  pressing  on  the  head,  to  gently  draw  the  head  away  from 
the  thorax,  so  that  by  this  expression  and  traction  movement  you 
extract  them  with  the  head  segment.  They  are  very  minute  and  are  to 
be  told  by  their  exceedingly  highly  refractile  appearance.  To  stain  for 
spcrozoites,  pick  up  the  head  end,  and  with  forceps  draw  the  severed 
neck  along  a  clean  dry  slide,  trying  at  the  same  time  to  smear  out  the 
adherent  salivary  glands.  After  drying,  stain  with  Wright's  stain. 
The  sporozoites  are  narrow  falciform  bodies  about  12  p  in  length, 
with  a  central  chromatin  dot. 

A  matter  about  which  there  is  dispute  is  as  to  whether  the  salivary 
glands  communicate  with  the  alimentary  canal.  Theobald  states 
that  there  is  no  connection  between  them. 

DIFFERENTIATION  OF  CULICIN^:  AND  ANOPHELIN^:. 

It  is  impossible  even  for  an  entomologist  to  differentiate  mos- 
quitoes without  recourse  to  elaborate  keys  and  tables.  It  is  a  com- 
paratively easy  matter,  however,  to  decide  as  to  whether  the  mosquito  is 
a  probable  malaria  transmitter  or  not. 

While  certain  characteristics  of  the  male  are  used  to  separate  the 
^dinae  from  other  subfamilies,  yet  it  is  only  with  the  female  that  we 
•concern  ourselves  in  differentiating  the  Culicinae  from  the  Anophelinac. 
Therefore,  it  is  first  necessary  to  distinguish  the  male  from  the  female. 
If  the  antennae  have  not  been  torn  off,  this  can  be  decided  by  the 
highly-adorned  plumose  antennae  of  the  male,  those  of  the  female  being 
sparsely  decorated  with  short  hairs.  The  palpi  of  the  Anophelinae  tend 
to  be  clubbed,  while  those  of  the  Culex  are  straight.  If  the  antennae 
have  been  broken  off,  look  for  the  claspers  at  the  end  of  the  abdomen. 


MOSQUITOES. 


247 


Having  determined  that  the  insect  is  a  female,  we  then  proceed  to  place 
it  either  in  the  subfamily  Culicinae  or  Anophelinae  by  a  study  of  the 
relative  length  of  the  palpi  to  the  proboscis.  If  the  palpi  are  shorter 
than  the  proboscis,  it  belongs  to  the  Culicinae;  if  as  long  or  longer,  to 
the  Anophelinae.  The  palpi  of  the  female  Megarhininae  are  also 
long,  but  the  proboscis  is  curved. 

Having  settled  on  the  subfamily,  we  separate  the  genera  by  con- 
sidering such  points  as  character  and  distribution  of  scales  on  back  of 
head,  wings,  thorax  and  abdomen;  banding  of  proboscis,  legs,  ab- 
domen and  thorax,  shape  of  scales  on  wings  and  location  of  cross-veins. 

In  the  resting  position  Culex  allows  the  abdomen  to  droop,  so  that 


FIG.  78. — Anopheles.  FIG.  79. — Culex. 

Resting  positions  of  anopheles  and  culex  insects.  (Drawn  by  C.  O.  Waterhouse.} 


it  is  parallel  to  the  wall.     The  angle  formed  by  the  abdomen  with  head 
and  proboscis  gives  a  hunchback  appearance. 

Anopheles  when  resting  on  a  wall  goes  out  in  a  straight  line  at  an 
angle  of  about  45°.     It  resembles  a  bradawl. 

Classification. 

There  are  4  subfamilies  of  Culicidae,  differentiated  according  to 
the  palpi: 


i.  Palpi  as  long  or  longer 
than  proboscis  in  male. 


1.  Palpi  as  long  as  proboscis  in  female;  proboscis 
straight.     Anophelina. 

2.  Palpi  as  long  or  shorter  than  proboscis;  probos- 
cis curved.     Megarrhinince. 

3.  Palpi  shorter  than  proboscis.     Cuticince. 
2.  Palpi  shorter  than  proboscis  in  male  and  female.     JEdince. 


248 


THE    MOSQUITOES. 


The  important  ones  from  a  medical  stand-point  are  the  Anophelin 33 
and  Culicinae. 


i.  Scales  on  head  only; 
hairs  on  thorax  and 
abdomen. 


2.  Scales  on  head  and 
thorax  (narrow  curved 
scales.)  Abdomen  with 
hairs. 


Anophelinae. 

1.  Scales  on  wings,   large  and   lanceolate.     Ano- 
pheles. 

2.  Wing  scales  small  and  narrow  and  lanceolate. 
Myzomyia. 

3.  Large   inflated   wing   scales.     Cyclolcppteron. 

i.  Wing  scales  small  and  lanceolate.     Pyretophorus. 


i.  Abdominal    scales    only    on    ventral    surface. 
Thoracic  scales  like  hairs.     M yzorhynchus . 


3.  Scales  on  head  and 
thorax  and  abdomen. 
Palpi  covered  with 
thick  scales. 


2.  Abdominal    scales    narrow,  curved    or  spindle- 
shaped.     Abdominal  scales  as  tufts  and  dorsal 
patches.     Nyssorhynchus. 


3.  Abdomen  almost  completely  covered  with  scales 
and  also  having  lateral  tufts.     Cellia. 


4.  Abdomen  completely  scaled.     Aldrichia. 


NOTE. — Of  the  above  genera  only  Cycloleppteron  and  Aldrichia  are  unproven 
malarial  transmitters. 

The  Megarhininae  are  of  no  importance  medically. 
The  genus  Megarhinus  has  the  following  characteristics : 

1.  Large  mosquitoes  with  brilliant  metallic  coloring.     (Ele- 
phant mosquitoes.) 

2.  Long,  curved  proboscis. 

3.  Caudal  tufts  of  hairs  on  each  side  of  abdomen. 

The  JEd'msd  are  not  known  to  play  any  role  in  transmission  of 
diseases.  This  subfamily  is  characterized  by  having  the  maxillary 
palpi  much  shorter  in  both  males  and  females  than  the  proboscis. 

One  genus  Sabethes  is  very  characteristic,  owing  to  dense  paddle- 
like  scale  structures  on  two  or  more  legs. 


MOSQUITOES.  249 

Differentiation  of  Culicinae  Genera. 

i.  Posterior  cross-          i.  Proboscis  curved   in  female.     Psorophora. 
vein     nearer    the          2.  Proboscis  straight  in  female, 
base  of  the  wing  A.  Palps  with  3  segments  in  the  female. 


than  the  midcross- 


vein. 


a.  Third  segment  somewhat  longer  than  the 
first  two.     Culex. 

b.  The  3  segments  equal  in  length.    Stegomyia. 

B.  Palps  with  4  segments  in  the  female. 

a.  Palps  shorter  than  the  third  of  the  proboscis. 
Spotted  wings.     Theobaldia. 

b.  Palps  longer  than  the  third  of  the  proboscis. 
Irregular  scales  on  wings.     Mansonia. 

C.  Palps  with  5  segments  in  the  female.      Tcenior- 
hynchus. 


2.  Posterior  cross-vein  in  line  with  midcross-vein.     Joblotina. 

3.  Posterior  cross-vein  further  from  base  of  wing  than  midcross-vein.  Mucidus. 

Of  the  Culicinae  the  genus  Stegomyia  is  of  importance  on  account 
of  yellow  fever.  The  totally  efficient  hosts  for  filariasis  (filarial 
embryos  found  in  thorax  and  proboscis)  are  chiefly  among  the  genus 
Culex.  The  genera  Mansonia  and  Taeniorhynchus  may  also  trans- 
mit filariasis.  Some  think  the  Anophelinae  genera  "Cellia"  and 
"Myzomyia"  may  transmit  filariasis  as  well  as  malaria. 

The  genus  Culex  is  implicated  in  dengue. 

Stegomyia. — This  is  the  most  important  culicine  genus.  These 
are  mosquitoes  with  silver  markings.  The  head,  entirely  covered 
with  flat  scales,  has  also  some  upright  forked  scales.  Scutellum  has 
dense  flat  scales.  S.  Calopus  is  deep  blackish-brown  with  two  thoracic 
parallel  lines  with  curved  silver-white  lines  outside.  Banding  of 
thorax,  abdomen  and  legs. 

Culex. — Male  palpi  long  and  acuminate.  Head  has  narrow 
curved  and  upright  forked  scales.  Laterally,  flat  scales.  C.  fatigans 
supposed  to  carry  dengue. 

Theobaldia. — These  Culicinae  have  spotted  wings  resembling 
Anophelinae.  These  spots  are  due  to  aggregations  of  scales,  not  to 
dark  scales.  Male  palps  are  clubbed  (like  Anopheles). 

Mucidus. — This  genus  has  a  mouldy  look  from  long  twisted  gray 
scales. 

Mansonia. — This  genus  is  characterized  by  broad  flat  asym- 
metrical wing  scales. 


250  THE    MOSQUITOES. 

Grabhamia. — Wings  have  pepper-and-salt  appearance  with  short 
fork  cells. 

Tceniorhynchus. — This  genus  is  characterized  by  dense  wing 
scales,  which  are  broadly  elongated  with  truncated  apex. 

Acartomyia. — Much  like  Grabhamia,  but  scales  of  head  give  ragged 
appearance.  A.  zammittii  was  supposed  to  be  concerned  in  Malta 
fever  (not  proven). 


NOTES  ON  ANIMAL  PARASITOLOGY. 


NOTES  ON  ANIMAL  PARASITOLOGY. 


NOTES  ON  ANIMAL  PARASITOLOGY. 


NOTES  ON  ANIMAL  PARASITOLOGY. 


PART  IV. 

CLINICAL    BACTERIOLOGY    AND    ANIMAL    PARASITOL- 
OGY  OF  THE  VARIOUS  BODY  FLUIDS  AND  ORGANS. 


CHAPTER  XXII. 

Diagnosis  of  Infections  of  the  Ocular  Region. 

IT  is  advisable  before  taking  material  for  cultures  or  smears  to 
cleanse  the  nasal  area  of  the  eye-lids,  and  especially  about  the  carun- 
cles, with  sterile  salt  solution.  Then,  by  gently  pressing  on  the  lids,  we 
may  be  able  to  get  pure  cultures  of  the  organism  causing  the  infection. 
Normally,  we  may  find  in  the  region  of  the  caruncles  various  skin 
organisms,  especially  staphylococci,  giving  white  colonies. 

A  small  particle  of  sterile  cotton,  wound  on  a  toothpick,  with  the 
aid  of  a  sterile  forceps,  makes  an  excellent  swab  for  obtaining  material 
for  smears;  the  same  may  first  be  drawn  over  an  agar  surface  in  a 
Petri  dish  in  a  series  of  parallel  lines  of  inoculation  before  making  the 
smears  on  slide  or  cover-glass. 

When  there  is  considerable  discharge,  a  capillary  pipette,  with  a 
rubber  bulb,  may  be  used  to  draw  up  sufficient  material  for  cultures 
and  smear.  Be  sure  to  round  off  the  end  of  the  pipette  in  the  flame 
and  not  to  use  a  very  fine  capillary  tube. 

In  conjunctival  tultures,  plates  of  glycerin  agar  or  agar  plates 
smeared  with  blood  are  to  be  preferred,  as  the  gonococcus  and  Koch- 
Weeks  bacillus  will  only  grow  on  blood  or  hydrocele  agar.  The 
diphtheria  and  xerosis  bacilli  grow  well  on  glycerin  agar. 

In  addition  to  the  white  staphylococcus,  the  streptococcus  may  be 
present  when  inflammation  of  the  nasal  duct  exists. 

The  pneumococcus  is  a  fairly  common  cause  of  serpiginous  corneal 
ulcerations.  Active  treatment  is  necessary. 

251 


252  DIAGNOSIS    OF    INFECTIONS    OF    THE    OCULAR    REGION. 

The  B.  xerosis  is  possibly  a  harmless  organism  and  must  not  be 
accepted  as  explaining  an  infection  unless  other  factors  have  been 
eliminated. 

Leprosy  and  tubercle  bacilli  may  be  found  in  corneal  ulcerations. 
The  true  diphtheria  bacillus,  which  the  xerosis  so  much  resembles, 
may  cause  a  pseudomembranous  inflammation. 

The  gonococcus  and  the  Koch-Weeks  bacillus  are  usually  re- 
sponsible for  the  very  acute  cases  of  conjunctivitis.  Both  these 
organisms  are  characteristically  intracellular  and  are  Gram  negative. 

The  diplobacillus  of  Morax  and  Axenfeld  is  more  common  in 
chronic,  rather  dry  affections  of  the  conjunctiva. 

In  cases  of  ozena  with  involvement  of  the  nasal  ducts  Fried- 
lander's  bacillus  may  be  found. 

Certain  fungi  of  the  genus  Microsporum  have  been  thought  to  be 
the  cause  of  trachoma,  as  have  also  certain  bacillary  forms.  One 
should  be  very  conservative  about  reporting  fungi  in  smears  or  cultures 
of  external  surfaces. 

The  larval  stage  of  Taenia  solium  (Cysticercus  cellulosae)  has  a 
predilection  for  eye  as  well  as  brain.  It  is  usually  situated  beneath 
the  retina. 

The  adult  Filaria  loa  tends  at  times  to  appear  under  the  conjunc- 
tiva or  in  the  subcutaneous  tissue  of  the  eye-lids. 

Fly  larvae  have  been  reported  from  the  conjunctival  sacs  in  the 
helpless  sick 


CHAPTER  XXIII. 
DIAGNOSIS  OF  INFECTIONS  OF  THE  NASAL  CAVITIES. 

IN  taking  material  from  the  nasal  cavities,  for  the  bacteriological 
examination,  it  is  well  to  wash  about  the  alae  with  sterile  water  and 
then  have  the  patient  blow  his  nose  on  a  piece  of  sterile  gauze  and 
take  the  material  for  culture  or  smear  from  this.  If  the  material  is 
purulent  and  located  at  some  ulcerating  spot,  it  is  best  to  use  a  specu- 
lum, and  either  touch  the  spot  with  a  sterile  swab  or  use  a  capillary 
bulb  pipette  with  a  slight  bend  at  the  end. 

Normally,  we  find  only  white  staphylococcus  colonies  and  colonies  of 
short-chain  streptococci.  The  M.  tetragenus,  B.  xerosis  and  Hoff- 
man's bacillus  are  also  occasionally  found. 

In  some  cases  of  ozena  we  may  find  an  organism  of  the  Fried- 
lander  type  in  pure  culture. 

Biscuit-shaped  diplococci,  both  Gram  negative  and  positive,  are 
to  be  found  either  normally  or  in  cases  of  coryza.  M.  catarrhalis  has 
probably  been  frequently  reported  as  the  meningococcus.  Still,  the 
meningococcus  has  been  found  in  the  nasal  secretion  of  patients  with 
cerebrospinal  meningitis. 

Diphtheria  involving  the  nasal  cavity  must  always  be  kept  in  mind, 
and  in  quarantine  investigations  the  examination  of  the  nasal  secretion 
culturally  should  be  a  part  of  the  routine. 

The  tubercle  bacillus  may  be  found  in  nasal  ulcerations ;  it  is,  how- 
ever, only  present  in  exceedingly  small  numbers.  On  the  other  hand, 
one  of  the  best  diagnosistic  procedures  in  leprosy  is  to  examine  smears 
from  nasal  mucous  membrane  for  the  B.  leprae.  In  such  ulcerations 
the  bacilli  are  found  in  the  greatest  profusion.  •. '  M 

The  gonococcus  has  been  reported  for  the  nose.  Various  fungi 
have  been  reported  from  the  nose,  but  in  such  a  region  the  strictest 
conservatism  in  reporting  should  be  observed. 

Recently  sporozoa  have  been  reported  in  a  case  of  nasal  polyp. 

253 


254  DIAGNOSIS    OF    INFECTIONS    OF    THE    NASAL    CAVITIES. 

So  many  degenerative  changes  in  epithelial  cells  resemble  protozoal 
forms  that  such  findings  require  ample  confirmation. 

The  larval  form  of  the  Linguatula  rhinaria  is  a  rare  parasite  of  the 
nasal  cavities. 

Various  fly  larvae  are  far  more  common,  and  the  "screw-worm," 
the  larva  of  the  Chrysomyia  macellaria,  is  common  in  certain  parts 
of  tropical  America,  and  may  by  its  burrowing  effects  cause  fatal 
results. 


CHAPTER  XXIV. 
EXAMINATION  OF  BUCCAL  AND  PHARYNGEAL  MATERIAL. 

IN  a  preparation  made  from  material  taken  by  a  sterile  swab  from 
the  region  of  the  normal  buccal  and  pharyngeal  cavities  and  stained  by 
Gram's  method,  we  are  struck  by  the  variety  of  organisms  present. 

Gram  positive  and  Gram  negative  staphylococci  are  present,  as  are 
also  streptococci,  pneumococci,  leptothrix  forms  and  very  probably 
yeasts  and  sarcinae  types  with  many  Gram  negative  bacilli.  If 
pseudodiphtheria  organisms  are  present,  we  have  these  showing  a 


FIG.  80. — Vincent's  angina.     Spirochaeta  vincenti.     (Coplin.) 


Gram  positive  reaction.  If  this  material  is  smeared  on  agar  plates 
and  cultured  at  37°  C.,  we  are  struck  by  the  fact  that  the  colonies  on  the 
plates  may  be  exclusively  staphylococcal  and  streptococcal. 

It  is  very  difficult,  if  not  impossible,  to  distinguish  a  pneumo- 
coccus  colony  from  a  streptococcus  one  on  a  plate  culture.  The  presence 
or  absence,  however,  of  the  pneumococcus  is  distinctly  shown  in  the 

255 


256         EXAMINATION    OF    BUCCAL    AND    PHARYNGEAL    MATERIAL. 

Gram  stained  smear,  either  by  its  lance-shaped  morphology  or  the 
presence  of  a  capsule.  In  has  been  my  experience  that  smears  from 
about  15%  of  normal  individuals  show  capsulated  pneumococci. 

In  diphtheria  examinations  we  rely  chiefly  on  the  cultural  findings  on 
Loffler's  serum.  Where  the  process  is  streptococcal  or  due  to  the 
organisms  associated  with  Vincent's  angina,  the  immediate  examina- 
tion of  a  smear  from  the  suspected  spot  or  area  gives  greater  diagnostic 
information.  The  streptococcus  being  so  abundant  in  cultures  from 
normal  throats,  it  is  difficult  to  determine  its  significance  in  a  culture ; 
abundance  of  streptococci  in  a  smear  from  an  ulceration  or  bit  of 
membrane,  however,  is  of  etiological  import. 

By  staining  with  Neisser's  method  it  is  possible  to  make  an  im- 
mediate diagnosis  of  diphtheria  from  a  smear  from  a  piece  of  mem- 
brane in  about  25  percent  of  cases.  It  is  well,  however,  to  always 
culture  such  material. 

Material  from  the  throat  is  ordinarily  best  obtained  with  a  sterile 
copper  wire  cotton  pledget  swab.  The  platinum  loop  usually  bends 
too  easily.  A  sterile  forceps  may  be  more  convenient  for  obtaining 
particles  of  membrane.  It  is  believed  that  ulcerative  conditions  of  the 
throat,  associated  with  the  presence  of  the  large  fusiform  bacillus  and 
delicate  spirillum,  which  make  the  picture  of  Vincent's  angina,  are 
more  common  than  is  usually  so  considered.  As  a  rule,  only  cultures 
on  serum  are  made  and  very  rarely  direct  smears.  If  a  smear  were 
always  made  and  stained  by  Gram's  method  (with  a  contrast  stain  of 
dilute  carbol  fuchsin)  at  the  same  time  the  culture  was  made,  it  is 
probable  that  much  information  of  value  would  be  obtained. 

Direct  smears  are  the  procedure  of  choice  in  streptococcal  and 
pneumococcal  anginas  as  well  as  in  Vincent's  angina. 

Unless  very  familiar  with  the  morphology  of  Treponema  pallidum 
and  using  Giemsa's  staining  procedure,  we  should  be  very  conserva- 
tive in  reporting  such  an  organism  from  suspected  syphilitic  ulcera- 
tions  of  the  throat. 

The  thrush  fungus  (Endomyces  albicans)  may  be  easily  demon- 
strated in  a  Gram  stained  specimen  as  violet  mycelial  structures. 

Yeasts  due  to  food  particles  are  not  infrequently  observed  in  smears 
and  cultures  from  the  mouth. 


ANIMAL   PARASITES    OF   THE   THROAT.  257 

Amoebae  and  flagellates  have  been  reported  from  the  mouth. 
Also  in  the  remarkable  disease  "halzoun,"  flukes  have  been  found  to 
be  the  cause  of  the  asphyxia. 

In  the  tropics,  round  worms  may  be  vomited  up  and,  lodging  in  the 
pharynx,  may  have  to  be  extracted. 


CHAPTER  XXV. 
EXAMINATION  OF  SPUTUM. 

FREQUENTLY  the  material  submitted  for  examination  as  sputum  is 
simply  buccal  or  pharyngeal  secretion,  or  more  probably  secretion 
from  the  nasopharynx,  which  has  been  secured  by  hawking.  It 
should  always  be  insisted  upon  that  the  sputum  be  raised  by  a  true 
pulmonary  coughing  act,  and  not  expelled  with  the  hacking  cough  so 
frequently  associated  with  an  elongated  uvula.  When  there  is  an 
effort  to  deceive,  some  information  may  be  obtained  from  the  watery, 
stringy,  mucoid  character  of  the  buccopharyngeal  material  and  also, 
from  the  presence  of  mosaic-like  groups  of  flat  epithelial  cells  (often 
packed  with  bacteria).  The  pulmonary  secretion  is  either  frothy 
mucus  or  mucopurulent  material,  and  if  the  cells  are  alveolar  they 
greatly  resemble  the  plasma  cells.  At  times  these  cells  may  contain 
blood  pigment  granules  (heart  disease  cells). 

In  the  microscopic  examination  a  small,  cheesy  particle,  the  size  of  a 
pin  head,  should  be  selected.  This  should  be  flattened  out  in  a  thin 
layer  between  the  slide  and  cover-glass  and  should  be  examined  for 
elastic  tissue,  heart-disease  cells,  eggs  of  animal  parasites,  amoebae  and 
fungi.  Echinococcus  booklets,  Curschman  spirals  besprinkled  with 
Charcot-Leyden  crystals,  and  haematoidin  and  fatty  acid  crystals  may 
also  be  observed. 

It  may  facilitate  the  examination  of  the  sputum  for  elastic  tissue 
and  actinomycosis  and  other  fungi  to  add  10%  sodium  hydrate  to  the 
preparation. 

To  make  smears  for  staining,  the  sputum  should  be  poured  on  a 
flat  surface,  preferably  a  Petri  dish,  and  a  bit  of  mucopurulent 
material  selected  with  forceps.  A  dark  back-ground  facilitates  picking 
out  the  particle.  A  toothpick  is  well  adapted  to  smearing  out  such 
material  on  a  slide.  After  using,  it  can  be  burned.  When  dry,  the 
smear  is  best  fixed  by  pouring  on  a  few  drops  of  alcohol,  allowing  this 

258 


TUBERCLE    BACILLI  IN   SPUTUM.  259 

to  run  over  the  surface,  and  then,  after  dashing  off  the  excess  of 
alcohol,  to  ignite  that  remaining  on  the  film  in  the  flame  and  allow  to 
burn  out. 

A  mark  with  a  grease  pencil,  about  one-half  inch  from  the  end, 
gives  a  convenient  surface  to  hold  with  the  forceps  and  also  prevents 
the  stain  subsequently  used  from  running  over  the  entire  surface. 
Sputum  should  as  a  routine  measure  be  stained  by  the  Ziehl-Neelson 
method  and  by  Gram's  method. 

In  examining  for  tubercle  bacilli  it  may  be  necessary  to  employ 
some  method  for  concentrating  the  bacterial  content  of  the  sputum 
prior  to  making  the  smear.  A  very  satisfactory  method  is  that  of 
Muhlhauser-Czaplewski.  Shake  up  the  sputum  with  four  to  eight 
times  its  volume  of  1/4%  solution  of  sodium  hydrate  in  a  stoppered 
bottle.  When  the  mixture  has  become  a  smooth,  mucilaginous -looking 
fluid,  add  a  few  drops  of  phenolphthalein  solution  and  bring  the  pink 
mixture  to  a  boil. 

Then  add  drop  by  drop  a  2%  solution  of  acetic  acid,  stirring  con- 
stantly, until  the  pink  color  is  just  discharged.  If  the  least  excess  of 
acid  is  added  over  that  just  sufficient  to  cause  the  pink  color  to  disap- 
pear, mucin  wrill  be  precipitated.  Now  pour  this  mixture  into  a  centri- 
fuge tube  and  smear  the  sediment  on  a  slide  and  stain  for  tubercle 
bacilli. 

Sputum  smears  stained  by  some  Romanowsky  method  or  by  the 
haematoxylin  eosin  stain  are  best  adapted  for  the  study  of  various 
cells,  and  in  particular  of  the  eosinophile  cells  so  characteristic  of 
bronchial  asthma.  In  sputum  from  cancer  of  the  lungs  the  large 
vacuolated  cells  may  be  found. 

When  examining  the  sputum  of  the  bronchopneumonia  of  in- 
fluenza the  formol  fuchsin  gives  the  best  results.  The  influenza 
bacilli  are  found  in  little  masses,  frequently  grouped  about  small 
collections  of  M.  tetragenus.  The  cocci  stain  a  rich  purplish-red, 
while  the  small  influenza  bacilli  take  on  a  light  pink  color. 

Red  cells  show  up  well  in  specimens  stained  by  the  Romanowsky 
method;  if  rouleaux  formation  is  marked,  it  may  indicate  pulmonary 
infarction. 

In  culturing  sputum  a  mucopurulent  mass  should  be  washed  in 


260  EXAMINATION    OF    SPUTUM. 

sterile  water  and  should  then  be  dropped  into  a  tube  of  sterile  bouillon. 
With  a  sterile  swab  it  should  be  emulsified  and  successive  streaks  made 
along  the  surface  of  an  agar  or  a  glycerin  agar  plate.  In  obtaining 
cultures  from  influenza  sputum,  first  smear  the  material  thoroughly 
over  a  blood-serum  slant;  then  inoculate,  by  thorough  smearing  over 
the  surface  of  successive  blood-streaked  agar  slants,  the  material  on 
the  surface  of  the  blood- serum  slant.  The  platinum  loop  should  be 
transferred  from  one  slant  to  another  without  recharging.  The  in- 
fluenza bacillus  seems  to  grow  better  if  the  blood-streaked  agar  slants 
are  prepared  just  before  inoculating  with  the  sputum.  All  that  is 
necessary  is  to  sterilize  an  ear,  puncture  and  take  up  the  exuding 
blood  with  a  large  loop.  Cultures  for  tubercle  bacilli  are  impracti- 
cable. A  guinea-pig  should  be  inoculated. 

The  blood-stained  watery  sputum  of  plague  pneumonia  should  be 
cultured  on  plates  of  plain  agar  and  3%  salt  agar  at  the  same  time. 
An  ordinary  smear  stained  with  carbol  thionin,  however,  practically 
makes  a  diagnosis. 

Pneumococci,  M.  catarrhalis,  and  Friedlander's  bacillus  in  sputum 
are  best  demonstrated  by  Gram's  method  of  staining. 

Moulds,  especially  Aspergilli,  may  be  found  in  sputum.  Species 
of  Mucor,  Cryptococcus  and  Endomyces  have  also  been  reported. 

Amoebae  from  liver  abscess  rupturing  into  the  lung  may  be  found. 
Very  important  pulmonary  infections  are  those  with  Paragonimus 
westermani.  This  is  recognized  by  the  presence  of  operculated  eggs  in 
the  sputum. 

Hydatid  cysts,  either  of  the  lung  or  of  the  liver,  rupturing  into  the 
lung,  may  be  recognized  by  the  presence  of  echinococcus  hooklets. 
The  material  is  bile-stained  if  from  the  liver. 


CHAPTER  XXVI. 
THE  URINE. 

MATERIAL  for  staining  is  best  obtained  by  centrifuging  the  urine, 
then  pouring  off  the  supernatant  urine,  to  replace  it  with  a  i%  aqueous 
solution  of  formalin.  Shaking  for  a  few  seconds  we  again  centrifuge, 
pour  off  the  fluid  from  the  sediment  and  make  smears  from  the  sedi- 
ment. The  smear  may  be  stained  directly  by  Wright's  method  or 
after  fixing  by  heat  with  Gram's  stain,  T.  B.  stain  or  haematoxylin  and 
eosin.  The  latter  is  the  best  for  the  staining  of  epithelial  cells  and 
animal  parasites;  the  Gram  method  for  bacteria. 

It  is  frequently  difficult  to  distinguish  the  spores  of  moulds  from 
red  blood-cells  except  by  measurement  and  staining  reactions.  Spores 
of  moulds  rarely  exceed  five  mikrons. 

It  is  difficult  to  determine  the  presence  of  blood  in  urine  in  higher 
dilution  than  i  to  300  with  the  spectroscope.  The  occult  blood-test 
will  show  it  in  much  higher  dilution. 

To  secure  urine  for  bacteriological  examination  catheterization 
is  rarely  necessary.  The  glans  penis  and  meatus  should  be  thoroughly 
washed  with  soap  and  water,  after  which  dilute  alcohol  (50%)  should 
be  used.  The  greater  part  of  the  urine  first  passed  should  be  rejected 
and  only  the  last  portion  passed  should  be  caught  in  a  sterile  recep- 
tacle. This  may  be  either  streaked  over  the  surface  of  an  agar  or  a 
lactose  litmus  agar  plate.  This  lattter  medium  is  very  useful  in 
distinguishing  typhoid  or  paratyphoid  colonies  (blue)  from  colon,  and 
streptococcus  or  staphylococcus  colonies  (pink).  The  urine  may  be 
added  to  tubes  of  melted  agar  and  then  poured. 

Cystitis  from  a  colon  infection  gives  an  acid  urine;  that  caused 
by  Proteus  vulgaris  an  alkaline  urine. 

The  bacilli  of  plague  and  Malta  fever  are  also  found  in  the  urine. 

\Yhile  the  smegma  bacillus  in  urine  may  be  differentiated  from  the 

tubercle  bacillus  by  the  former  losing  its  red  color,  by  prolonged 

decolorization  with  acid  alcohol,  yet  it  is  chiefly  by  the  subcutaneous 

inoculation  of  the  guinea-pig  that  we  should  diagnose  genitourinary 

18  261 


262 


THE    URINE. 


tuberculosis.  Inject  the  sediment  after  centrifuging.  Gonococci  are 
reported  from  Gram  stained  smears. 

Yeasts  and  moulds  frequently  contaminate  urine,  especially  diabetic 
urine,  after  it  has  been  passed.  Amoebae  and  flagellates  (Trichomo- 
nas  vaginalis  in  females)  may  be  found  in  urine.  Also  the  itch  mite 
is  not  rarely  found  in  the  urine  of  those  having  scabies  of  the  penis. 

Eggs  of   Schistomum   haematobium    (bilharziosis)  are  important 


0 


fl-H-EV^ 


ti  .9 - 


FIG.  81. — Starches  and  fibres  found  in  urine. 

diagnostic  findings;  these  are  terminal-spined.  Those  of  rectal 
bilharziosis  are,  as  a  rule,  lateral-spined. 

In  chylous  urine  the  filarial  embryos  may  be  found.  This  examina- 
tion is  facilitated  by  centrifugalization. 

The  eggs  of  the  E.  gigas  may  be  recognized  in  urinary  sediment 
by  their  pitted  appearance. 

The  vinegar  eel  may  be  found  in  the  urine  of  females  who  have 
used  vaginal  douches  of  vinegar. 


CHAPTER  XXVII. 
THE  FAECES. 

IF  the  fecal  examination  is  to  be  made  for  the  diagnosis  of  amoebae, 
in  a  case  where  the  characteristic  mucous  stools  are  not  present,  or  to 
verify  the  existence  of  flagellates,  it  is  best  to  give  a  dose  of  salts  early 
in  the  morning  and  examine  the  liquid  stools  which  follow  such  treat- 
ment. This  treatment  is  satisfactory  for  examination  for  intestinal 
parasites  and  ova. 

The  blood-flecked  mucus  of  bacillary  dysentery  or  a  piece  of 
mucus  from  a  typical  amoebic  dysentery  stool  are  best  suited  for 
cultural  examination. 

If  the  purpose  of  the  examination  is  to  determine  the  digestive 
power  of  the  alimentary  tract  for  proteids,  carbohydrates  or  fats,  it  is 
best  to  use  a  test  diet,  as  that  of  Schmidt  and  Strasburger. 

Prior  to  using  this  test  diet,  one  should  familiarize  himself  with 
the  microscopic  appearances  resulting  from  such  a  diet  in  a  normal 
person;  information  is  then  at  hand  to  judge  of  variations  from  the 
normal.  The  examination  of  the  faeces  7)f  persons,  on  ordinary  and 
specifically  undetermined  articles  of  diet,  is  very  unsatisfactory  when 
the  state  of  digestion  of  muscle  fibers  and  the  question  of  fat  digestion 
is  at  issue. 

In  examining  the  faeces  of  the  normal  person  and  likewise  with  the 
patient,  wait  until  the  second  or  third  day  so  that  the  faeces  of  previous 
diets  may  have  passed  out. 

Diet:  breakfast,  7  A.  M.,  bowl  of  oatmeal  gruel  (40  grams  oatmeal, 
10  grams  butter,  200  c.c.  milk,  300  c.c.  water).  Also  one  very  soft- 
boiled  egg  (i  min.)  and  50  grams  zwieback.  In  the  forenoon,  500  c.c. 
of  milk. 

For  dinner,  2  o'clock,  chopped  beef  broiled  very  rare  (125  grams 
with  20  grams  butter  poured  over  it.)  Also  a  potato  puree  (200  grams 

263 


264 


THE    FAECES. 


mashed  potato,  50  grams  milk,  10  grams  butter).  Also  one-half 
liter  of  milk  and  50  grams  zwieback. 

For  supper,  7  o'clock,  the  same  articles  as  for  breakfast. 

Having  familiarized  one's  self  with  the  degree  of  digestion  of 
muscle,  starch  and  fat  in  a  normal  person,  we  are  in  a  position  to  judge 
of  the  state  of  assimilation  in  a  patient. 

We  judge  of  muscle  digestion  by  the  intactness  of  the  striations. 


FIG.  82. — Familiar  objects  in  feces.  i,  muscle  fibres;  2,  soaps;  3,  vegetable 
hairs;  4,  fatty  acid  crystals  projecting  from  neutral  fat  globule;  5,  soap  crystals 
with  leukocytes;  6,  stone  cells;  7,  vegetable  spirals;  8,  pallisade  cells  from  bean; 
9,  parenchyma  of  vegetable  tissue;  10,  u,  12  and  14,  vegetable  cells;  13,  pollen. 


If  a  muscle  remnant  is  only  a  homogeneous  yellowish  particle,  it  shows 
satisfactory  digestion.  If  it  is  rectangular,  with  well-defined  cross 
striations,  it  shows  poor  digestion  for  meat.  A  loopful  of  faeces  should 
be  smeared  into  a  drop  of  Lugol's  solution  for  starch-digestion  determi- 
nation. Normally  there  should  be  no  blue-staining  starch  granules 


CULT  I  RINC.    FAECES.  265 

Soaps  are  gnarled  bodies  everted  like  the  pinna  of  an  ear,  while 
soap  crystals  are  comparatively  coarse  and  do  not  melt  on  application 
of  gentle  heat  as  do  the  more  delicate  fatty  acid  crystals.  Neutral 
fat  is  in  round  or  irregular  globules.  The  best  stain  for  fat  is  Sudan 
III  (saturated  solution  of  Sudan  III  in  equal  parts  of  70%  alcohol 
and  acetone). 

Mix  up  the  faeces  with  dilute  alcohol  (50  to  70%)  and  then  add  a 
drop  of  the  above  solution  and  apply  a  cover-glass  quickly.  The  fat 
globules  show  as  orange  or  golden-yellow  bodies.  Much  connective 
tissue  debris  shows  defect  in  gastric  digestion,  as  only  the  stomach 
digests  connective  tissue. 

In  examining  a  liquid  stool  after  salts,  it  is  well  to  color  the  drop  of 
faeces,  which  is  to  be  covered  with  the  cover-glass,  with  a  small  loopful 
of  1/2%  solution  of  neutral  red.  If  diluting  fluid  is  used,  it  should  be 
salt  solution,  and  not  water.  The  neutral  red  tinges  the  granules  of 
the  endoplasm  of  amoebae  and  flagellates  a  very  striking  brown-red  color, 
thus  differentiating  them  from  vegetable  cells  or  body  cells. 

Whether  examining  the  thin  faeces  or  the  mucus  particle,  it  is  well 
to  reserve  report  on  amoebae  or  flagellates  until  motion  is  observed. 
Encysted  protozoa  are  difficult  to  diagnose. 

When  a  smear  preparation  is  desired,  we  may  smear  out  a  fragment 
of  mucus  and  stain  by  Romanowsky's  or  Gram's  method.  The  charac- 
ter of  the  bacteria  present  appears  to  be  of  diagnostic  value — especially 
in  the  case  of  infants  and  young  children.  Beautiful  preparations  may 
be  made  by  mixing  the  faeces  with  water,  then  centrifuging  for  one 
minute.  This  throws  down  vegetable  debris  and  crystals.  Now 
decant  the  supernatant  fluid,  which  holds  the  bacteria  in  suspension, 
and  add  an  equal  amount  of  alcohol.  Again  centrifuge,  decant,  and 
smear  out  and  examine  the  bacterial  sediment.  Gram's  method,  with 
dilute  carbol  fuchsin  counterstaining,  gives  the  best  picture. 

To  culture  for  typhoid,  dysentery,  cholera  or  other  bacteria,  take 
up  the  material  in  a  tube  of  sterile  bouillon  and  smear  it  out  with  a 
swab  over  a  lactose  litmus  agar  plate  or  an  Endo  or  Conradi-Drigal- 
ski  plate.  Before  streaking  the  plates  they  should  be  very  dry  on  the 
surface.  This  can  be  best  done  by  pouring  into  a  plate  with  a  circular 
piece  of  filter-paper  in  the  lid  and  placing  in  the  incubator  for  one-half 


266  THE    F^CES. 

hour  to  dry.  The  filter-paper  absorbs  the  moisture.  Then  inoculate 
the  surface  of  the  plate  with  the  fecal  material. 

Muller's  method  for  pancreatic  functioning  determination  is  to 
give  a  colomel  purge  two  hours  after  a  meal.  A  little  of  the  liquid 
stool  is  smeared  on  the  surface  of  blood-serum  and  the  tube  incubated 
at  60°  C.  (paraffin  oven).  If  the  surface  is  smooth,  no  trypsin  was 
present;  if  dotted  with  spots  of  digestion  liquefaction,  it  shows  that  the 
pancreatic  secretion  is  present.  This  should  be  tried  with  the  Cam- 
midge  reaction  of  the  urine. 

Epithelial  cells  are  generally  more  or  less  disintegrated.  In  the 
mucus  of  bacillary  dysenteric  stools,  however,  large  intact  phagocytic 
cells  are  frequent,  which  may  be  mistaken  for  encysted  amcebae. 

Triple  phosphate  crystals  are  frequently  observed,  as  may  also  be 
crystals  of  various  calcium  salts.  Charcot-Leyden  crystals  are  rather 
indicative  of  helminthiases. 

Various  flagellates,  and  in  particular  Lamblia,  may  be  responsible 
for  diarrhceal  conditions  which  may  cause  rather  serious  symptoms. 

Balantidium  coli  has  been  reported  several  times  as  the  cause  of 
dysenteric  conditions.  Coccidiadea  are  found  in  the  faeces. 

In  the  Philippines  the  Entamceba  histolytica  is  the  most  important 
of  the  animal  infections.  Besides  examining  for  it  in  a  cover-glass 
preparation,  we  should  attempt  to  make  cultures.  A  diluted  bouillon, 
i :  10,  containing  i  1/2%  agar,  with  a  reaction  about — 1.5,  is  a  satisfac- 
tory medium.  The  most  important  points  in  success  seem  to  be  proper 
symbiosis  and  proper  reaction.  In  a  series  of  four  tubes  containing 
the  same  media,  but  with  — 2.5,  — 2,  — 1.5,  and  — i,  the  amcebae  may 
only  grow  in  one  of  the  tubes.  Walker  recommends  smearing  a  cover- 
glass  with  suitable  agar,  then  inoculating  the  surface,  we  invert  it 
over  the  hollow  of  a  concave  slide,  sealing  the  margins  with  vaselin. 
This  enables  us  to  study  development  with  a  high  power  of  the 
microscope. 

It  is  in  the  faeces  we  examine  either  for  the  parasites  or  for  their  ova 
in  connection  with  practically  all  the  flukes,  except  the  lung  fluke  and 
the  bladder  fluke;  for  intestinal  tseniases  and  for  practically  all  the 
round  worms,  except  the  filarial  ones. 

In  the  tropics,  the  examination  of  the  faeces  vastly  exceeds  in  value 


F^CES.  267 

that  of  the  urine  and  is  possibly  more  important  than  blood  examina- 
tions. 

The  larvae  of  various  insects  may  at  times  be  detected  in  the  stools, 
as  well  as  certain  acarines  (cheese  mites,  etc.) . 

The  test  for  occult  blood  is  indicated  in  helminthiases  as  well  as  in 
the  conditions  for  which  it  is  usually  tested. 


CHAPTER  XXVIII. 
BLOOD  CULTURES  AND  BLOOD  PARASITES. 

CLINICALLY,  the  most  important  examination  of  the  blood  for  para- 
sites is  for  the  presence  of  various  bacterial  infections  and  for  certain 
blood  protozoa  and  also  nlarial  embryos. 

The  modern  method  of  culturing  blood,  especially  for  the  detection 
of  typhoid  or  paratyphoid  bacilli,  is  by  the  use  of  the-  bile  media  of 
Conradi.  Test-tubes  are  filled  with  7  to  10  c.c.  of  i%  peptone  ox  bile, 
or  ox  bile  alone,  and  the  medium  is  sterilized  in  the  autoclave.  It  is 
good  practice  to  place  the  syringe  in  a  plugged  test-tube  containing 
salt  solution,  with  the  needle  unscrewed.  After  autoclaving,  the 
sterile  syringe  can  be  taken  to  the  bedside  in  the  test-tube.  Using  a 
wide  test-tube,  a  forceps  can  be  sterilized  at  the  same  time  and  used  to 
attach  the  needle  to  the  barrel  of  the  syringe. 

The  skin  should  be  scrubbed  gently  with  green-soap  solution  and 
water  for  about  three  minutes.  The  skin  of  the  area  to  be  punctured 
should  then  be  sterilized  by  the  gentle  application  of  Harrington's 
solution  (riot  scrubbed)  for  one-half  minute,  and  should  then  be  washed 
with  sterile  water.  It  appears  to  be  safe  to  simply  scrub  the  area  with 
70%  alcohol  for  one  or  two  minutes.  A  tourniquet  is  now  applied  to 
distend  the  vein,  and  the  needle  is  inserted  in  the  direction  of  the  venous 
flow.  Withdrawing  5  to  10  c.c.  of  blood,  we  loosen  the  tourniquet, 
then  withdraw  the  needle  (otherwise  the  blood  may  flow  from  the 
puncture),  and  force  out  about  1/2  c.c.  into  the  first  bile  tube,  about 
i  c.c.  into  the  second,  and  2  or  3  c.c.  into  the  third.  It  is  well  to 
reserve  some  of  the  blood  for  Widal  tests. 

The  bile  tubes  are  now  incubated  for  10  to  12  hours  and  then 
transfers  are  made  to  bouillon  tubes.  These  bouillon  tubes  can  be 
used  in  six  to  eight  hours  for  testing  the  organism  against  known 
typhoid  or  paratyphoid  sera. 

Some  prefer  to  streak  plates  of  lactose  litmus  agar  with  material 

268 


BLOOD   CULTURE.  269 

from  the  bile  tubes  instead  of  inoculating  the  bouillon  tubes.  Con- 
tamination with  staphylococci  or  the  presence  of  staphylococci, 
streptococci  or  plague  bacilli  in  septicaemic  conditions  show  easily 
accessible  colonies. 

Schotmuller  adds  i  to  3  c.c.  of  blood  to  liquefied  agar  at  45° 
C.,  and  after  mixing  pours  into  plates.  The  standard  method  for- 
merly was  to  add  the  blood  to  an  excess  of  bouillon  (i  to  5  c.c.  of  blood 
to  100  c.c.  or  more  of  bouillon).  By  using  the  bile  media,  we  can  take 
the  blood  from  the  ear  in  typhoid  cases,  if  preferred.  Then  if  chance 
staphylococcic  contamination  occurs,  such  colonies  are  readily  differen- 
tiated from  typhoid  ones  by  the  pink  color  on  lactose  litmus  agar.  In 
culturing  blood  in  septicaemic  conditions,  the  blood  should  always  be 
drawn  from  the  vein. 

Typhoid  cultures  are  best  obtained  in  the  first  week  of  the  disease, 
after  that  time  the  Widal  is  the  test  of  preference. 

If  a  paratyphoid  serum  is  not  at  hand  for  testing,  it  may  suffice  to 
inoculate  a  glucose  bouillon  tube;  gas  production  indicates  para- 
typhoid. This  test  should  be  applied  when  a  very  motile  organism  does 
not  show  agglutination  with  a  known  typhoid  serum.  Anthrax  and 
glanders  should  be  considered  in  blood  cultures. 

In  Malta  fever  it  must  be  remembered  that  colonies  do  not  show 
themselves  for  several  days.  Addition  of  blood  to  melted  agar  is  a 
good  procedure. 

The  examination  of  the  blood  for  the  parasites  of  malaria,  filariases, 
kala-azar  and  spirillum  fevers  has  been  discussed  under  their  respective 
headings. 

Trypanosomes  from  human  trypanosomiasis  have  not  as  yet  been 
cultured,  and  smears  from  gland  juice  or  cerebrospinal  fluid,  seem 
more  satisfactorv  to  examine  than  blood  smears. 


CHAPTER  XXIX. 
THE  STOMACH  CONTENTS. 

FROM  a  microscopical  stand-point  there  is  comparatively  little  that 
is  of  value  in  the  examination  of  the  gastric  contents ;  there  is  nothing 
very  specific  about  the  findings. 

A  test  meal  is  not  a  necessity  as  in  the  chemical  examination,  but 
either  vomitus  or  material  withdrawn  with  a  stomach-tube  two  or  more 
hours  after  an  ordinary  meal  suffice. 

The  microscopical  diagnostic  points  in  connections  with  distin- 
guishing cancer  of  the  stomach  from  nonmalignant  dilatation  are:  (i) 
Fragments  of  cancer  tissue.  These  are  very  rarely  found  and  are 
most  difficult  to  diagnose.  (2)  The  presence  of  flagellates  in  the  early 
stages  of  cancer  (the  so-called  anacid  stage  preceding  the  development 
of  lactic  acid).  As  flagellates  prefer  an  alkaline  medium,  they  disap- 
pear after  the  acidity  due  to  lactic  acid  comes  on.  (3)  The  presence  of  the 
Boas-Oppler  bacillus.  There  are  probably  several  organisms  so 
designated.  They  are  lactic  acid  producers  and  are  characterized  by 
being  very  large  bacilli  (7x1/1)  and  arranged  in  long  chains  which 
stretch  across  the  field  of  the  microscope.  They  are  Gram  positive 
and  do  not  form  spores.  They  can  be  cultivated  on  media  rich  in 
blood  and  are  aerobic.  They  should  only  be  reported  when  present 
in  great  abundance  and  in  long  chains.  (4)  The  absence  of  sarcinae 
and  yeasts.  The  presence  of  these  cocci  and  fungi  in  vomitus  is 
indicative  of  a  simple  dilatation. 


270 


CHAPTER  XXX. 
EXAMINATION  OF  PUS. 

Pus  may  be  collected  for  examination  either  (i)  with  a  platinum 
loop,  (2)  with  a  sterile  swab,  (3)  with  a  bacteriological  pipette  or  (4) 
with  a  hypodermic  syringe. 

It  is  always  well  to  make  a  smear  and  stain  it  by  Gram's  method  at 
the  same  time  that  cultures  are  made.  The  Gram  stain  gives  informa- 
tion as  to  the  abundance  of  organisms  in  the  pus  and  as  to  the  probable 
findings  in  the  culture.  Pneumococci  and  streptococci  are  differen- 
tiated from  the  staphylococci  in  this  way  without  the  necessity  of  more 
or  less  extended  cultural  methods. 

When  autogenous  vaccines  are  to  be  made,  the  isolation  of  the 
exciting  organism  is  necessary.  This  is  best  done  by  streaking  the 
pus,  taken  up  with  a  sterile  swab  and  emulsified  in  a  tube  of  bouillon, 
over  the  surface  of  an  agar  plate.  Practically  as  convenient  and  provid- 
ing a  more  nutritious  medium  is  to  smear  the  material  on  a  loop  or 
swab  over  the  surface  of  a  blood-serum  slant,  then  to  inoculate  a  second 
tube  from  the  first  without  recharging  the  loop  or  swab,  and  so  on  until 
three  or  four  tubes  are  inoculated.  Isolated  colonies  should  be  ob- 
tained in  the  third  or  fourth  tube. 

In  examining  blood  serum  slants  inoculated  with  purulent  material, 
always  examine  the  water  of  condensation  for  streptococci. 

A  bacteriological  pipette  is  very  useful  when  pus  is  to  be  sent  to 
a  laboratory;  the  tip  can  be  sealed  in  a  flame  and  the  cotton  plug  at 
the  other  end  insures  the  noncontamination  of  the  contents.  The 
material  may  be  drawn  up  either  with  the  mouth  or  with  a  rubber  bulb. 

The  hypodermic  syringe  is  very  useful  in  puncturing  buboes,  etc., 
especially  in  plague.  A  small  pledget  of  cotton  on  a  toothpick  dipped 
into  pure  carbolic  acid  and  touched  to  a  spot  over  the  bubo,  which 
after  about  thirty  seconds  is  soaked  with  alcohol,  makes  a  sterile 
anaesthetic  spot  at  which  to  introduce  the  needle  of  the  syringe.  It 

271 


272  EXAMINATION    OF    PUS. 

must  be  remembered  that  when  plague  buboes  begin  to  soften,  the 
plague  bacilli  may  be  replaced  by  ordinary  pus  organisms. 

It  is  remarkable  how  frequently  we  get  pure  cultures  from  abscess 
material.  In  purulent  material  from  abdominal  abscesses  we  are  apt 
to  obtain  mixed  cultures,  especially  the  colon  bacillus  and  B.  pyocy- 
aneus,  in  addition  to  ordinary  pus  organisms. 

When  it  is  a  question  between  streptococci  and  pneumococci,  it  is 
well  to  inoculate  a  mouse;  the  capsulated  pneumococci  at  the  autopsy 
make  the  diagnosis. 

Animal  inoculation  is  also  necessary  in  plague  and  glanders,  and 
possibly  anthrax.  When  tetanus  is  suspected,  it  should  be  examined  for 
as  described  under  Tetanus.  Tuberculosis  should  also  be  identified  by 
inoculating  a  guinea-pig,  as  well  as  by  acid-fast  staining  and  culture,  if 
there  be  any  doubt  as  to  the  nature  of  the  material. 

The  black  or  yellow  granules  of  madura  foot,  as  well  as  those  of 
actinomycosis,  should  be  examined  as  recommended  in  the  section  on 
fungi. 

Amoebae,  coccidia  and  larval  echinococci  may  be  found  in  purulent 
material,  as  may  also  various  other  animal  parasites,  as  fly  larvae, 
sarcopsyllae,  etc. 


CHAPTER  XXXI. 
SKIN   INFECTIONS. 

CULTURAL  methods  are  to  be  preferred  in  the  bacteriological 
examination  of  the  skin. 

This  is  best  done  by  washing  the  surface  to  be  examined  with  soap 
and  water,  in  order  to  eliminate  chance  organisms  which  may  have 
settled  on  the  surface  of  the  skin  in  dust  or  as  a  result  of  contact  with 
material  containing  them.  Scrapings  are  then  made  with  a  sterile  dull 
scalpel,  and  this  material  is  emulsified  in  a  drop  of  sterile  water  in  the 
center  of  a  Petri  dish.  A  tube  of  melted  agar  at  42°  C.  is  then  poured 
on  the  inoculated  drop  and,  by  mixing,  the  bacterial  flora  is  distributed 
over  the  entire  surface  of  the  plate.  Of  the  colonies  developing  on 
such  plates  probably  80%  will  be  found  to  be  staphylococci,  and  of 
these  the  greater  proportion  will  be  staphylococci  showing  white 
colonies. 

Occasionally  the  aureus  or  citreus  may  be  isolated. 

Streptococci  and  colon  bacilli  are  rarely  found. 

The  Staphylococcus  pyogenes  aureus  is  the  organism  usually 
isolated  from  furuncles,  circumscribed  abscesses  and  carbuncles. 

Streptococci  are  the  organisms  to  be  expected  in  phlegmonous  in- 
fections. 

Cold  abscesses,  which  are  frequently  due  to  tuberculous  infection, 
are,  as  a  rule,  sterile. 

Acne  pustules  may  show  staphylococci  or  the  microbacillus  of 
acne  may  be  present. 

The  bottle  bacillus,  which  morphologically  resembles  a  yeast,  is 
considered  to  be  the  cause  of  dry  pityriasis  capitis.  It  may  also  be  found 
in  the  comedones  of  children. 

In  the  tropics,  an  organism  which  at  times  produces  lesions  similar 
to  impetigo  and  again  pemphigoid  eruptions  and  at  other  times  wide- 
spreading  erysipelatous  conditions  gives  cultural  characteristics 

273 


274  SKIN    INFECTIONS. 

similar  to  S.  pyogenes  aureus.  It  is  probably  only  a  virulent  aureus. 
It  has  been  described  under  the  name  of  Diplococcus  pemphigi  con- 
tagiosi. 

The  Staphylococcus  epidermidis  albus,  or  stitch  abscess  coccus,  is 
considered  by  Sabouraud  to  be  the  cause  of  eczema  seborrhoicum. 

It  is  in  scrapings  from  the  skin  of  lepromata  that  we  find  acid-fast 
organisms  in  the  greatest  profusion.  In  tuberculosis  of  the  skin  the 
tubercle  bacilli  are  exceedingly  scarce.  Inoculation  of  a  guinea-pig 
will  probably  give  positive  results  with  the  tubercle  bacillus.  •  The 
leprosy  bacillus  is  noncultivable  and  noninoculable  for  experimental 
animals. 

Anthrax  and  glanders  cause  skin  lesions  which  can  only  be  surely 
diagnosed  culturally  or  by  animal  inoculation. 

Plague  bacilli  may  be  isolated  from  the  primary  vesicles  appearing 
at  the  site  of  the  flea  bite. 

Tropical  phagedaena  is  thought  by  some  to  be  due  to  a  sort  of 
diphtheroid  organism. 

The  skin  diseases  due  to  fungi  are  discussed  under  that  section. 
Of  the  skin  affections  caused  by  animal  parasites,  ground  itch  is  the 
most  important.  This  is  a  form  of  dermatitis  due  to  the  irritation  set 
up  by  the  hook-worm  larvae  penetrating  the  skin  of  the  foot  and  leg. 

The  Sarcopsylla  penetrans  or  jigger  (sand  flea)  is  an  important  agent 
in  ulcerations  about  the  foot. 

Certain  acarines  cause  skin  lesions,  as  is  also  the  case  with  the  larvae 
of  certain  flies. 

The  itch  mite  (Sarcoptes  scabiei)  is  an  important  animal  parasite 
of  the  skin. 

The  various  lice,  fleas  and  bed-bugs  are  well  understood  as  causes 
of  skin  irritation. 

Filarial  infections  are  also  important. 


CHAPTER  XXXII. 
CYTODIAGNOSIS. 

THIS  method  of  diagnosis  is  chiefly  employed  in  the  examination  of 
cellular  sediments  of  pleural,  ascitic  and  cerebrospinal  fluid. 

For  pleural  fluids  we  should  receive  the  material  in  centrifuge  tubes 
about  one-fourth  filled  with  2%  sodium  citrate  salt  solution.  This 
prevents  clotting.  Having  thrown  down  the  sediment,  the  superna- 
tant fluid  is  poured  off,  and  in  its  place  a  i%  aqueous  solution  of 
formalin  is  added.  After  mixing  and  allowing  to  stand  for  about  five 
minutes,  centrifugalization  is  again  repeated  and,  pouring  off  the 
supernatant  formalin  solution,  we  make  smears  from  the  sediment. 
This  is  either  stained  by  a  Romanowsky  method  or,  after  fixing  with 
heat  (burning  alcohol),  the  smear  is  stained  with  haematoxylin  and 
eosin. 

At  the  time  of  securing  fluid  for  cytodiagnosis,  cultures  should  be 
made  on  blood-serum  for  various  pyoqjenic  bacteria  and,  if  tuberculosis 
is  suspected,  inoculation  of  a  guinea-pig  is  indicated. 

The  interpretation  of  cellular  sediments  is  more  difficult  than  many 
books  would  indicate,  there  being  many  factors  which  tend  to  com- 
plicate the  findings.  The  following  are  the  leading  differentiations: 

1.  A  smear  showing  almost  entirely  lymphocytes  with  a  few  red 
cells  and  very  rarely    a  polymorphonuclear    indicates    a   tubercular 
process. 

2.  Where  a  pyogenic  process  is  engrafted  on  a  tuberculous  one,  we 
have  still  the  red  cells,  some  degenerated  lymphocytes  and  in  particular 
polymorphonuclears  showing  fragmentation  of  their  nuclei. 

3.  When  a   hydrothorax  results   from   chronic   heart  or  kidney 
disease,  the  characteristic  cell  is  the  endothelial  cell,  which  greatly 
resembles  a  large  mononuclear. 

4.  Some  authorities  consider  that  the  cancer  cell  can  be  recognized 
by  its  occurring  in  masses  and  having  a  markedly  vacuolated  cytoplasm. 

275 


276  CYTODIAGNOSIS. 

It  has  been  claimed  that  they  contain  glycogen  by  which  means  we  can 
distinguish  them  from  endothelial  cells  which  they  so  much  resemble. 

Jousset  introduced  ionoscopy  as  a  means  of  diagnosing  tuberculo- 
sis. The  fluid  was  allowed  to  coagulate  and  was  then  digested  with  an 
artificial  gastric  juice.  The  digested  material  was  then  centrifuged 
and  the  sediment  examined  for  tubercle  bacilli.  This  process  does  not 
seem  to  have  met  with  much  favor  in  this  country.  (Using  sodium 
citrate  obviates  the  necessity  for  digesting  the  coagulum.) 

The  same  points  will  hold  for  ascitic  fluid  as  for  pleural  fluid. 

In  taking  cerebrospinal  fluid  for  culture  and  cytodiagnosis  we  use 
a  stout  antitoxin  needle  without  attaching  a  syringe.  Aspiration  is 
responsible  for  many  of  the  ill  effects  of  lumbar  puncture.  The  needle 
should  be  about  four  inches  long  for  an  adult.  Sterilize  the  skin  and 
needle  as  described  for  blood  cultures  from  a  vein.  To  make  a 
lumbar  puncture,  place  patient  on  left  side  with  knees  drawn  up.  A 
line  at  the  level  of  the  iliac  crests  passes  between  the  third  and  fourth 
lumbar  vertebrae.  Select  a  point  midway  between  the  spinous  proc- 
esses of  these  lumbar  vertebrae  and  enter  the  needle  2/5  of  an  inch  to 
the  right  of  this  point,  pushing  the  needle  inward  and  upward.  Collect 
the  material  in  a  sterile  test-tube.  Make  cultures  on  blood-serum  and 
then  centrifugalize  and  examine  the  sediment  as  for  pleural  fluids. 

In  general  terms  it  may  be  stated  that: 

1.  A  lymphocytosis  indicates  a  tuberculous  process. 

2.  An  abundance  of  polymorphonuclear  and  eosinophilic  leuko- 
cytes indicates  a  meningococcic  or  pneumococcic  infection. 

A  method  of  examination  considered  by  neurologists  as  of  differen- 
tial diagnostic  value  is  to  count  the  number  of  cells  in  a  cubic  milli- 
meter of  the  cerebrospinal  fluid.  The  technic  is  to  use  a  gentian-violet- 
tinged  3%  solution  of  acetic  acid.  This  is  drawn  up  to  the  mark  0.5, 
and  the  cerebrospinal  fluid  is  then  sucked  up  to  n.  After  mixing,  the 
cell  count  is  made  with  the  haemocytometer.  Normally  we  have  only 
one  or  two  cells  per  cubic  millimeter,  but  in  tabes  or  general  paresis 
this  is  increased  to  50  or  100  cells. 

Trypanosomiasis  gives  a  cellular  increase  very  similar  to  syphilis. 


CHAPTER  XXXIII. 
RABIES. 

THIS  is  a  disease  of  dogs  and  wolves,  but  is  communicable  to  man 
and  domesticated  animals.  The  virus,  whatever  it  may  be,  resides  in 
the  saliva  and  nervous  structures.  It  is  destroyed  by  a  temperature  of 
50°  C.  In  man  the  period  of  incubation  is  usually  from  three  weeks  to 
three  months,  but  may  be  shorter  or  may  extend  over  one  year. 

Bites  about  the  face  and  those  with  marked  lacerations  are  particu- 
larly serious.  Bites  of  rabid  wolves  give  about  four  times  as  great 
a  mortality  as  those  of  dogs.  In  the  dog  there  are  two  types  of  the 
disease — dumb  rabies  and  furious  rabies. 

By  inoculating  rabbits  subdurally  with  an  emulsion  of  the  brain  or 
spinal  cord  of  a  rabid  animal,  and  successively  the  medulla  of  this 
rabbit  subdurally  into  other  rabbits,  we  finally  so  increase  the  virulence 
of  the  infection  that  rabbits  die  in  six  days.  Beyond  this  it  is  im- 
possible to  increase  the  virulence  and  it  is  termed  "fixed  virus."  To 
attenuate  this  virus  the  spinal  cord  of  the  rabbit  is  removed  and  is  dried 
over  caustic  potash.  The  cord  is  divided  into  segments  about  one 
inch  in  length.  Drying  for  about  fifteen  days  seems  to  entirely  de- 
stroy the  virus. 

To  prepare  the  material  for  prophylactic  injections  a  small  portion 
of  the  cord  is  emulsified  and  injected  subcutaneously.  The  German 
method  is  to  commence  with  a  cord  that  has  been  dessiccated  only  eight 
days.  At  first  injections  are  given  daily,  and  it  is  possible  to  inject 
three  days'  cords  by  the  sixth  day. 

The  treatment  lasts  for  about  twenty  days.  In  the  diagnosis  of 
rabies  in  dogs  it  is  preferable  to  preserve  the  animal  so  that  the  develop- 
ment of  the  symptoms  may  be  observed.  In  case  the  dog  has  been 
killed,  it  may  be  possible  to  make  a  diagnosis  by  means  of  the  Negri 
bodies.  These  are  round  or  oval  bodies  from  i  to  2o//  in  diameter, 
which  may  be  found  in  the  nerve-cells,  especially  those  of  the  cornu 
19  277 


278  RABIES.  . 

ammonis  (Hippocampus  major).  They  may  be  demonstrated  by 
staining  smears  of  brain  substance  by  some  Romanowsky  method, 
especially  by  the  Giemsa  stain.  As  their  relation  to  the  nerve-cell  is 
more  or  less  disturbed  by  such  a  method,  it  is  preferable  to  fix  brain 
tissue  about  the  region  of  the  cornu  ammonis  in  Zenker's  fluid,  then  to 
imbed  in  paraffin  and  make  sections.  These  are  stained  with  Giemsa 's 
stain  and  the  Negri  bodies  are  brought  out  as  lilac-red  bodies  in  the 
blue  cytoplasm  of  the  nerve-cells.  It  is  necessary  to  differentiate  in 
95%  alcohol.  In  addition  to  examining  for  the  Negri  bodies,  a  rabbit 
may  be  inoculated  subdurally  with  a  sterile  salt-solution  emulsion  of 
the  medulla  of  the  dead  dog. 

If  the  brain  or  cord  of  the  dog  are  to  be  sent  to  a  laboratory  for 
examination  they  should  be  packed  in  ice  or  placed  in  glycerin.  Take 
of  glycerin  one  part  and  one  part  water.  Sterilize  the  diluted  glycerin 
by  boiling,  allow  to  cool,  and  drop  the  pieces  of  brain  tissue  into  this. 
This  does  not  kill  the  virus.  It  is  supposed  by  some  that  Negri  bodies 
are  protozoal  in  nature  notwithstanding  the  fact  that  the  virus  will  pass 
through  a  coarse  Berkefeld  filter. 

Antirabic  serum  has  been  prepared  by  injecting  sheep  with  emul- 
sions of  rabid  rabbits'  cord  and  brain — at  first  intravenously,  then 
subcutaneously. 


MISCELLANEOUS  NOTES. 


MISCELLANEOUS  NOTES. 


MISCELLANEOUS  NOTES. 


MISCELLANEOUS  NOTES. 


APPENDIX. 

A— PREPARATION  OF  TISSUES  FOR  EXAMINATION  IN  MICROSCOPIC 

SECTIONS. 

i.  Fixation: 

a.  It  is  most  important  that  the  tissues  to  be  examined  be  placed  in  the  fixing 
fluid  as  soon  after  death  or  operation  as  possible.     Degenerative  changes  are  in 
this  way  avoided. 

b.  The  piece  of  tissue  to  be  fixed  must  not  be  too  large.     Using  a  sharp  scalpel, 
or  preferably  a  razor,  a  slab  of  tissue  about  one-half  an  inch  square  and  not  more 
than  one-fifth  of  an  inch  thick  should  be  dropped  into  the  bottle  containing  the  fixa- 
tive.    The  bottom  of  this  bottle  should  have  a  thin  layer  of  cotton  with  a  piece  of 
filter-paper  covering  it.     There  should  be  at  least  twenty  times  as  great  a  volume 
of  fixing  fluid  as  of  tissue  to  be  fixed.     Delicate  tissues,  as  pieces  of  gut,  should 
be  attached  to  pieces  of  glass,  wood  or  cardboard. 

c.  The  most  convenient  fixative  for  the  average  medical  man  is  a  10%  solution 
of  ordinary  commercial  formalin  (4%  of  formic  aldehyde  gas),  either  in  water  or, 
preferably,  in  normal  salt  solution.     Fixation  is  complete  in  from  12  to  24  hours. 
By  placing  in  the  incubator,  at  37°  C.,  2  to  12  hours  in  the  formalin  solution  suffices. 
If  fixed  in  the  paraffin  oven  (56°  C.),  fixation  is  accomplished  in  about  one-half 
hour. 

Formalin  once  used  for  fixation  must  be  thrown  away. 

The  fixative  which  probably  gives  the  best  histological  pictures  and  with  which 
we  obtain  the  most  satisfactory  haematoxylin  staining  is  Zenker's  fluid.  This  is 
Muller's  fluid  containing  5%  of  corrosive  sublimate.  It  also  contains  5%  of  glacial 
acetic  acid,  which  latter  is  only  added  just  before  we  are  ready  to  fix  the  piece  of  tissue. 
Muller's  fluid  is: 

Pot.  bichromate,        2.5  grams. 
Sod.  sulphate,  i.o  grams. 

Water,  t     100.0  c.c. 

Zenker's  fluid  fixes  in  about  24  hours.  After  all  corrosive  sublimate  fixatives 
we  should  wash  the  tissues  in  running  water  for  12  to  24  hours.  The  precipitate 
of  mercury  in  the  tissues  is  best  gotten  rid  of  by  treating  the  section  on  the  slide  with 
Lugol's  solution,  rather  than  the  tissue  in  bulk  with  iodine  alcohol. 

In  Orth's  fluid  we  add  10%  of  formalin  to  Muller's  fluid  (recommended  for 
nerve  tissue). 

A  saturated  corrosive  sublimate  solution  in  salt  solution  with  the  addition  of  5% 
of  glacial  acetic  acid  may  be  used  as  a  substitute  for  Zenker's  fluid. 

279 


280  APPENDIX. 

2.  Dehydration. — After  washing  for  twelve  to  twenty-four  hours  in  running 
water,  following  corrosive  sublimate  fixation,  or  simply  "washing  for  a  few  minutes 
after  formalin,  the  tissues  should  be  placed  in  70%  alcohol.     They  may  be  kept- in 
this  indefinitely.     If  they  are  to  be  sent  to  a  laboratory  for  sectioning,  it  is  advisable 
lo  moisten  a  pledget  of  cotton  in  70%  alcohol  and  fill  in  the  bottom  of  the  bottle  with 
it.     Then  drop  in  the  tissues  and  pack  in  gently  over  them  sufficient  70%  alcohol 
saturated  cotton  to  fill  up  the  bottle.     All  the  alcohol  should  be  absorbed  by  the 
cotton  so  that  if  the  bottle  should  break  in  transit  there  would  be  no  damage  from 
the  alcohol.     The  stopper  of  the  bottle  should  be  paraffined  or  sealed  with  wax. 

Tissues  may  be  left  in  the  70%  alcohol  1 2  to  24  hours  and  should  then  be  trans- 
ferred to  95%  alcohol  for  an  equal  time.  They  are  then  transferred  to  absolute 
alcohol,  where  they  remain  from  2  to  12  hours  and  are  then  placed  in  xylol.  The 
time  in  xylol  should  be  as  short  as  possible.  So  soon  as  the  tissue  looks  clear  it 
should  be  removed — 30  minutes  to  two  hours. 

3.  Imbedding. — The  tissue  is  now  transferred  to  melted  paraffin.      Paraffin 
melting  at   48°  C.  for  winter  work,   and   that   melting   at   54°   C.   for  summer. 
The  time  in  the  paraffin  should  not  he  prolonged.     Two  hours  will  ordinarily  suffice. 
Some  leave  in  the  paraffin  for  12  to  24  hours. 

Next  take  a  paper  box  (made  of  stiff  writing-paper  folded  over  a  square  of  wood) 
and  fill  with  the  melted  paraffin.  As  quickly  as  possible  drop  in  the  piece  of  tissue 
taken  out  of  the  paraffin  bath  with  heated  forceps  and,  so  soon  as  the  paraffin  begins 
to  solidify  on  the  surface,  place  the  paper  box  in  ice  water.  When  paraffin  is  rapidly 
cooled,  crystallization  is  less. 

The  Acetone  Method. — Take  the  tissues  out  of  the  70%  alcohol  and  place  in 
acetone.  After  remaining  in  acetone  for  one  to  two  hours,  the  tissues  should  be 
transferred  to  fresh  acetone  for  an  equal  length  of  time.  They  should  then  be 
placed  in  xylol  for  about  one-half  hour  and  then  imbedded  in  paraffin  as  directed 
above. 

The  Chloroform  Method. — The  procedure  may  be  the  same  as  in  the  method  of 
passing  through  alcohols  to  xylol,  substituting  chloroform  for  xylol  and  then  trans- 
ferring to  paraffin. 

Where  absolute  alcohol  is  not  obtainable,  very  satisfactory  results  may  be  obtained 
by  transferring  to  a  mixture  of  95%  alcohol  and  chloroform  after  immersion  in  95% 
alcohol.  Then  going  from  the  alcohol-chloroform  mixture  to  pure  chloroform 
thence  to  paraffin. 

When  a  piece  of  tissue  is  not  more  than  one-fourth  inch  square  and  one-eighth 
inch  thick,  it  is  very  easy  to  run  it  through  in  three  to  six  hours.  Thus: 

10%  Formalin  (in  37°  C.  incubator),  i  hour. 

70%  Alcohol  (in  37°  C.  incubator),  i  hour. 

95%  Alcohol  (in  37°  C.  incubator),  i  hour. 

Absolute  Alcohol  (in  37°  C.  incubator),  1/2  hour. 

Xylol  (in  37°  C.  incubator),  1/2  hour. 

Paraffin  (in  55°  C.  incubator),  1/2  to  2  hours. 


APPENDIX.  28l 

It  is  preferable  to  have  a  good  microtome.  The  best  is  that  of  Minot.  Very 
satisfactory  sections  can  be  cut  with  the  various  types  of  student  microtomes,  costing 
from  $12  to  $20. 

(In  using  a  hand  microtome,  a  razor  with  a  flat  edge  is  necessary.  After  experience, 
sections  thin  enough  for  histological  but  not  for  bacteriological  examination  can  be 
made.) 

If  the  piece  of  tissue  is  properly  dehydrated  and  imbedded,  thin  sections  (3  to  IOM) 
should  be  easily  obtained,  provided  the  knife  be  sharp.  One  advantage  about  the 
paraffin  method  is  that  it  is  only  necessary  to  have  a  small  part  of  the  blade  in 
proper  condition.  With  celloidin  the  entire  cutting  edge  must  be  perfect.  Having 
cut  the  sections,  they  should  be  dropped  on  the  surface  of  a  bowl  of  warm  water 
(45°  C.).  This  causes  the  section  to  flatten  out  evenly. 

Decalcification. — This  is  best  accomplished  by  fixing  in  10%  formalin  for  24 
hours,  then  placing  a  small  piece  of  the  bone  (not  exceeding  one-half  inch  square 
and  one-fifth  of  an  inch  thick)  in  concentrated  sulphurous  acid. 

This  decalcifies  in  about  24  hours.  Wash  thoroughly  in  alkaline  water  and  then 
in  tap  water.  Pass  through  alcohols  and  xylol  and  imbed  and  section  as  before 
described. 

To  Stain  Sections. — It  is  first  necessary  to  affix  the  section  to  a  slide  or  cover- 
glass. 

To  attach  the  section  firmly  to  the  slide,  so  that  it  will  not  become  detached  in 
subsequent  treatment,  pick  up  a  section  on  a  strip  of  cigarette  paper. 

A  sheet  of  cigarette  paper  is  cut  into  about  five  pieces  (1/2  x  i  1/2  ins.). 
Inserting  the  strip  of  cigarette  paper  under  the  section,  it  is  easily  lifted  up  out  of 
the  water.  Then  apply  the  slip  of  cigarette  paper,  section  downward,  to  a  perfectly 
clean  slide.  Blot  with  a  piece  of  filter-paper,  then  strip  off  the  piece  of  filter-paper 
leaving  the  section  smoothly  applied  to  the  slide.  Next  place  in  the  37°  C.  incubator 
for  twelve  to  twenty-four  hours  and  the  section  will  be  found  to  be  so  firmly  attached 
that  it  will  not  be  dislodged  by  subsequent  treatment. 

For  Immediate  Diagnosis. — Take  a  loopful  of  albumin  fixative  (white  of  fresh 
egg,  50  c.c.;  glycerin,  50  c.c.;  sodium  salicylate,  i  gram)  and  deposit  it  on  a  cover- 
glass.  Now  take  up  a  loopful  of  30%  alcohol  (i  drop  of  95%  alcohol  and  two  drops 
of  water)  and  applying  it  over  the  albumin  fixative,  smear  out  the  mixture  uniformly 
over  the  cover-glass. 

2.  Pick  up  a  section  on  a  strip  of  cigarette  paper  and  apply  it  to  the  prepared  sur- 
face on  the  cover-glass.     Blot  with  gentle  pressure  with  a  piece  of  filter-paper  over 
the  strip  of  cigarette  paper,  and  strip  off  this  latter,  leaving  the  section  attached  to 
the  cover-glass. 

3.  Now,  turning  the  flame  of  theBunsen  burner  down  very  low  or  with  a  small 
alcohol  flame,  we  hold  the  cover-glass  in  a  Stewart's  forceps,  section  side  up,  over 
the  flame  and  slowly  lower  it  until  the  paraffin  is  observed  to  melt.     This  shows  a 
temperature  of  about  50°  C.     The  section  is  fixed  by  the  coagulation  of  the  albumin 
at  about  70°  C.     To  obtain  this  temperature  lower  the  cover-glass  still  more,  and  the 


282  APPENDIX. 

moment  vapor  is  seen  to  rise  from  the  section  it  indicates  the  attachment  of  the  section 
to  the  cover-glass. 

4.  Flood  section  on  cover-glass  or  slide  with  xylol;  this  dissolves  out  the  paraffin. 
It  is  better  to  pour  off  the  first  xylol  and  drop  on  fresh  xylol  (one  minute). 

5.  Remove  xylol  with  two  applications  of  absolute  alcohol  (one  minute). 

6.  Treat  specimen  with  two  or  three  applications  of  95%  alcohol   (one  to  two 
minutes). 

7.  Next  wash  in  water  (one  to  two  minutes). 

8.  Flood  specimen  with  haemalum  of  Delafield's  haematoxylin  (three  to  seven 
minutes). 

9.  Wash  in  tap  water  for  about  two  to  five   minutes  until  a  purplish  tinge  is 
developed  in  the  section.     The  alkali  in  ordinary  tap  water  develops  this  color. 

10.  Apply  i  to  1000  eosin  for  thirty  seconds  to  one  minute. 

11.  Wash  in  water;  then  in  95%  alcohol;  then  in  absolute  alcohol. 

1 2.  Apply  a  few  drops  of  xylol  and  as  soon  as  the  section  is  perfectly  transparent 
mount  in  balsam. 

The  staining  by  haematoxylin  and  eosin  is  the  best  for  the  study  of  the  histology 
of  a  section.  It  only  requires  about  ten  minutes  to  run  a  preparation  through  for 
diagnosis  by  this  method. 

The  reagents  are  best  kept  in  dropping-bottles. 

The  staining  of  sections  on  slides  is  exactly  as  for  those  on  cover-glasses.  Cop- 
lin's  staining  jars  are  very  convenient  for  use  in  staining  slides. 

Where  the  cover-glass  method  is  used,  staining  by  Gram's  method,  acid-fast  stain- 
ing, capsule  staining,  etc.,  may  be  carried  out  as  for  bacterial  preparations. 

For  staining  Gram  positive  bacteria  in  sections,  the  Gram  method  as  for  bacterial 
preparations,  using  dilute  carbol  fuchsin  as  a  counter  stain,  gives  good  results. 

For  Gram  negative  bacteria  stain  with  thionin  as  for  blood  preparations  (10  to  20 
minutes).  Then  differentiate  in  i  to  500  acetic  acid  solution  for  ten  to  twenty 
seconds,  wash  with  water,  then  with  95%  alcohol,  and  quickly  through  absolute 
alcohol  and  xylol. 

Nicolle's Method. — i.  Stain  with  Loffler's  methylen  blue  ten  to  fifteen  minutes. 

2.  Differentiate  in  i  to  500  acetic  acid  ten  to  twenty  seconds. 

3.  Place  in  i%  solution  of  tannin  for  a  few  seconds  (fixes  color). 

4.  Wash  in  water,  then  into  95%  alcohol,  absolute  alcohol,  xylol  and  balsam. 
Van  Giesen's  Stain.— Take  of  one  percent  aqueous  solution  acid  fushsin  from 

5  to  15  c.c.  Saturated  aqueous  solution  picric  acid  100  c.c.  The  method  of  using 
is  to  first  stain  with  hsematoxylin  in  the  usual  way.  Then  pour  on  the  picro-acid  fuch- 
sin solution  and  allow  to  stain  for  one  to  five  minutes.  Wash,  pass  through  alcohols, 
and  xylol  and  mount  in  balsam. 

Connective-tissue  fibres,  axis  cylinders  and  ganglion  cells  are  stained  a  bright 
garnet  red.  Myelin,  muscle  fibers  and  cells  generally  are  stained  yellow.  Nuclear 
staining  is  that  of  haematoxylin.  The  stronger  stain  is  used  for  nerve  tissue;  the 
weaker  for  demonstrating  connective  tissue  in  tumors. 


APPENDIX.  283 

Romanowsky. — Staining  sections  with  Romanowsky  stains  is  not  very  satis- 
factory. The  differential  staining  seems  to  fade  out  in  passing  through  the  alco- 
hols. This  may  be  avoided  by  blotting  the  section  after  staining  and  differentiation 
and  then  applying  the  xylol  to  the  blotted  section.  After  staining  with  Giemsa's 
stain  for  10  to  15  minutes,  differentiate  with  i  to  500  acetic  acid.  When  the  section 
has  a  pinkish  tinge,  wash  in  water,  dry,  clear  in  xylol  and  mount. 

A  very  satisfactory  Giemsa  may  be  made  by  taking  i  gram  of  methylene  blue, 
1/2  gram  of  sodium  bicarbonate  and  100  c.c.  of  water  and  polychroming  as  for  King's 
stain.  Remove  the  dried  stain  from  the  porcelain  dish  and  put  it  into  a  bottle. 
Then  rinse  out  the  dish  with  methyl  alcohol  and  pour  into  the  bottle.  There  should 
be  100  c.c.  for  this  amount  of  stain.  Let  stand  3  to  5  hours,  then  filter.  To  the  100 
c.c.  of  methyl-alcohol  solution  add  100  c.c.  of  glycerin  containing  1/2  gram  of 
yellow  eosin. 

B— MOUNTING  AND    PRESERVATION   OF  ANIMAL  PARASITES. 

To  Mount  Small  Round  Worms. — Wash  the  hook,  whip  or  filarial  worm  in 
salt  solution,  then  drop  in  70%  alcohol  containing  5%  of  glycerin;  the  glycerin- 
alcohol  mixture  being  at  a  temperature  of  60°  C.  When  cool,  pour  into  Petri  dishes 
and  allow  the  alcohol  to  evaporate  in  the  37°  C.  incubator. 

Mount  in  glycerin  jelly,  preferably  in  a  concave  slide,  and  ring  the  preparation 
with  gold  size.  The  following  is  the  formula  for  Kaiser's  glycerin  jelly:  Soak 
one  part  of  gelatin  in  6  parts  of  distilled  water  for  two  hours.  Then  add  7  parts 
of  glycerin.  To  the  mixture  add  i%  of  carbolic  acid,  warm  for  15  minutes,  with 
constant  stirring,  and  then  filter  through  cotton. 

To  Prepare  Tape-worms. — Wash  in  salt  solution.  Wrap  around  a  piece  of 
glass  as  a  glass  slide  and  fix  in  salt  solution  containing  2  to  5%  of  formalin. 
Then  keep  the  preparation  permanently  in  70%  alcohol.  If  preferred,  the  specimen 
may  be  run  through  alcohols  and  xylol  and  mounted  in  balsam. 

Larvae. — Mosquito  larvae  may  either  be  prepared  as  for  small  round  worms 
or  they  may  be  dropped  into  70%  alcohol  at  60°  C.  and  then  passed  through  alcohols 
and  cleared  in  xylol  and  mounted  in  balsam.  Flukes  and  insects  may  require 
treatment  with  hot  (60°  to  70°  C.)  solution  of  10  to  20%  sodium-hydrate  solution. 
Then  wash  thoroughly  in  water  and  subsequently  pass  through  alcohols  to  xylol 
and  mount  in  balsam.  Clove  oil  or  cedar  oil  clears  more  slowly,  but  makes 
specimens  less  brittle  than  does  xylol.  Another  satisfactory  method  is  to  drop 
insects  or  larvae  into  acetone  at  60°  C.  and  after  being  in  this  from  i  to  12  hours  to 
clear  in  xylol  or  clove  oil  and  mount  in  balsam. 

Looss  has  a  method  of  first  washing  a  small  nematode  or  delicate  fluke  in  salt 
solution.  Then  pouring  this  first  salt  solution  out  of  the  test-tube  in  which  the  wash- 
ing was  carried  out,  to  add  fresh  salt  solution,  and  then  an  equal  amount  of  saturated 
aqueous  solution  of  bichloride  of  mercury,  The  shaking  is  easily  carried  on  in  the 
test-tube.  After  washing  in  water  the  worm  is  passed  through  alcohols,  one  strength 
of  which  should  contain  iodine.  Clear  in  xylol  and  mount  in  balsam. 


284  APPENDIX. 

To  Prepare  Flies  or  Mosquitoes  for  Transmission  Through  the  Mails. — 

Wrap  the  insect  carefully  in  a  piece  of  tissue-paper  (toilet-paper  answers).  Impreg- 
nate sawdust  with  5%  carbolic  acid  solution  and  fill  around  the  folded  insects  in  the 
box  containing  them.  (Barely  moisten.) 

C— PREPARATION  OF  NORMAL  SOLUTIONS. 

A  normal  solution  is  one  which  contains  the  hydrogen  equivalent  of  an  element, 
expressed  in  grams,  dissolved  in  sufficient  distilled  water  to  make  1000  c.c.  The 
hydrogen  equivalent  is  the  atomic  weight  of  any  element  divided  by  its  valence. 
In  a  base,  salt  or  acid  we  use  the  molecular  weight  in  grams  divided  by  valence. 

What  may  be  considered  as  the  valence  of  a  base  is  shown  by  the  number  of 
hydroxyls  combined  with  it;  that  of  an  acid  by  the  number  of  replaceable  hydrogen 
atoms  which  it  contains. 

To  make  a  normal  solution,  dissolve  in  distilled  water  a  weight  in  grams  equal 
to  the  sum  of  the  atomic  weights  of  the  substance,  divided  by  its  valence,  and  make 
up  the  volume  to  exactly  1000  c.c. 

NaOH  is  univalent.  Na  =  23.  O  =  16.  H  =  i.  Dissolve  40  grams  NaOH 
in  water  and  make  up  to  exactly  1000  c.c. 

Oxalic  acid  is  COOH — COOH  +  2  H2O  which  gives  it  a  molecular  weight  of 
126.  As  it  contains  two  carboxyl  groups  it  is  dibasic,  and  it  is  necessary  to  divide 
the  molecular  weight  by  2,  so  that  for  a  normal  solution  of  oxalic  acid  we  dissolve 
63  grams  in  a  volume  of  distilled  water  made  up  to  1000  c.c. 

If  a  chemical  laboratory  is  not  accessible  one  may  prepare  normal  solutions  with 
an  error  so  slight  as  to  be  unimportant  in  clinical  work  in  the  following  way: 

Sodium  hydrate  being  very  hygroscopic,  it  is  impossible  to  accurately  prepare  a 
normal  solution  by  directly  weighing  out  the  substance.  Instead,  select  perfect 
crystals  of  oxalic  acid,  such  as  can  be  obtained  in  a  drug  store,  and  weigh  out  on 
the  most  accurate  apothecary  scales  obtainable  exactly  6.3  grams  of  the  most  per- 
fect crystals  in  the  bottle.  Put  these  preferably  in  a  volumetric  flask  and  make 
up  with  distilled  water  to  1000  c.c.  Less  accurate  is  the  use  of  a  measuring  cylinder. 
If  care  is  used  this  should  give  N/io  solution  of  oxalic  acid  in  which  the  error  is  less 
than  i%. 

Having  N/io  acid  at  hand,  we  may  prepare  N/io  NaOH  in  the  following  way: 
Weigh  out  an  excess  of  sodium  hydrate  (5  grams  of  stick  caustic  soda)  and  dissolve 
in  1 100  c.c.  of  distilled  water.  Take  up  xoc.c.  of  this  solution  with  a  pipette  and 
let  it  run  into  a  beaker.  Add  six  drops  of  phenolphthalein  solution.  This  gives  a 
violet-pink  color.  Fill  the  burette  with  the  N/io  oxalic-acid  solution  and  let  it  run 
into  the  sodium-hydrate  solution  in  the  beaker  until  the  pink  is  just  discharged. 
Reading  off  the  number  of  c.c.  of  the  N/io  acid  used,  we  know  the  strength  of  the 
sodium-hydrate  solution.  It  is  well  to  repeat  the  titration  and  take  an  average. 

If  10.5  c.c.  of  the  oxalic-acid  solution  were  required  it  would  show  that  the 
sodium-hydrate  solution  was  stronger  than  N/io,  as  only  10  c.c.  would  have  been 


APPENDIX.  285 

necessary  if  the  NaOH  solution  had  been  N/io.  It  is  therefore  necessary  to  dilute 
the  sodium-hydrate  solution  in  the  proportion  of  10  to  10.5.  Measure  exactly  1000 
c.c.  of  the  too  concentrated  sodium-hydrate  solution  and  add  to  it  50  c.c.  of  distilled 
water,  mix  thoroughly,  and  we  have  1050  c.c.  of  N/io  solution  of  NaOH.  1000  x 
10.5  =  10.500.  10.500-7-10=1.050. 

As  Acidum  hydrochloricum  U.  S.  P.  is  about  two-thirds  water  (68.1%)  to  make 
N/io  HC1,  which  would  require  3.65  in  1600  c.c.,  it  would  be  necessary  to  take  about 
three  times  this  amount  of  U.  S.  P.  acid.  Take  12  c.c.  of  the  acid  and  add  distilled 
water  to  make  1 100  c.c.  Put  10  c.c.  of  this  dilute  solution  in  a  beaker.  Add  phenol- 
phthalein  solution  and  titrate.  If  n  c.c.  of  N/io  NaOH  were  required  it  would  be 
necessary  to  add  100  c.c.  of  water  to  a  volume  of  1000  c.c.  of  the  diluted  hydrochloric 
acid.  looo  x  n  =  nooo-f-  10  =  noo. 

Other  acid  and  alkali  solutions  can  be  made  as  for  N/io  HC1  and  N/io  NaOH. 

D— DISEASES  OF  UNKNOWN  OR  NOT  DEFINITELY  DETERMINED 

ETIOLOGY. 

OF  TEMPERATE  CLIMATES. 

Acute  Articular  Rheumatism. — Various  bacteria  have  been  reported  as  cause. 

Foot-and-mouth  Disease. — Probably  due  to  an  ultramicroscopic  organism. 

Measles. — Cause  entirely  unknown.     Hektoen  has  shown  that  blood  contains 
.the  virus. 

Mumps. — Herb  has  implicated  a  diplococcus.  Inoculations  into  Steno's  duct 
of  monkey  successful. 

Rabies. — Probably  the  Negri  bodies. 

Roetheln  (German  Measles). — Nothing  known. 

Scarlet  Fever. — Streptococci  seem  most  probable  cause  (S.  anginosus). 
Mallory  has  implicated  epithelial  protozoa. 

Small-p3X  and  Vaccinia. — Guarnieri  and  Councilman  have  implicated  epithe- 
lial protozoa. 

Spotted  Fever  of  the  Rocky  Mountains. — Supposed  to  be  due  to  an  unknown 
protozoon  transmitted  by  a  tick. 

Typhus  Fever. — It  has  been  suggested  that  the  cause  may  be  a  protozoon  trans- 
mitted by  vermin. 

Varicella. — Entirely  unknown. 

Whooping  Cough. — Influenza-like  bacilli  have  been  implicated. 
OF  TROPICAL  CLIMATES. 

Ainhum. — (A  disease  characterized  by  a  constricting  fibrous  ring,  especially 
of  little  toe,  often  leading  to  spontaneous  amputation.) 

Beriberi. — Various  microorganisms  and  food  factors  suggested. 

Blackwater  Fever. — Considered  as  a  malarial  disease,  but  thought  by  some 
to  be  possibly  caused  by  a  protozoon — a  Babesia  (Piroplasma) 

Dengue. — Supposed  to  be  due  to  a  protozoon  transmitted  by  Culex  fatigans 


286  APPENDIX. 

Goundou. — (Symmetrical  bony  tumors  of  nasal  processes  of  superior  maxillary 
bones.) 

Sprue. — (A  form  of  chronic  diarrhoea  characterized  by  diaphanous  thinning 
of  gut  and  ulcerations  of  buccal  cavity.) 

Tsutsugamushi. — (A  disease  of  Japan  somewhat  resembling  typhus  fever.) 
Supposed  to  be  due  to  a  protozoon  transmitted  by  the  Kedani  mite. 

Yellow  Fever. — Supposed  to  be  due  to  a  protozoon  transmitted  by  the  Stego- 
myia  calopus. 

E— MINK'S  MODIFICATION  OF  UNNA'S  H^EMATOXYLIN. 

Hsematoxylin,  .  i  gram. 

Alum,  8  grams. 

Sulphur  (sublimed),  i  gram. 

Glycerin,  30  c.c. 

Alcohol,  50  c.c. 

Water,  100  c.c. 

Dissolve  the  haematoxylin  in  the  glycerin  in  a  mortar.  Dissolve  the  alum  in  the 
water  and  add  it  to  the  glycerin  baematoxylin  in  the  mortar.  Then  add  the 
sulphur  and  the  alcohol.  The  solution  ripens  in  about  3  to  4  days.  Allow  the 
sediment  to  remain  in  the  bottom  of  the  bottle  containing  the  stain  and  filter  off 
small  quantities  as  needed. 


INDEX. 


Abbe  condenser,  4 

Abscess,  bacteria  in,  272 

Acanthia  lectularia,  228,  230 

Acarina,  221 

Acartomyia,  250 

Acetone,  for  sections  (see  tissue),  280 

Acid-fast  bacteria,  66 

Acid-fast  staining,  30 

Actinomycosis  (see  Discomyces),   106, 

272 

^Edinae,  248 
Agar,  egg,  22 

glucose,  22 

glycerine,  22 

nutrient,  21 

plating,  34 

Agchylostoma  duodenale,  208,  216 
Agglutination,  macroscopical,  127 

microscopical,  126 
Ainhum,  286 

Air,  bacteriological  examination  of,  117 
Aldrichia,  248 
Alexin,  122 
Amboceptor,  121 
Amoebae,  175 
Anaemia,  aplastic,  156, 

pernicious,  154,  164 

primary,  164 

secondary,  166 
Anaerobes,  53',  56 

Buchner  method,  58 

cultivation  of,  57 

Liborious  method,  58 

Vignal  method,  59 

Wright  method,  59 
Anginas,  256 

Animal  parasites,  general  classification, 
169 

mounting  of,  283 

nomenclature  in,  171 

preservation  of,  283 
Ankylostoma,  208 
Anophelinae,  246,  248 


Anthrax,  53,  54 

vaccination,  56 
Antitoxin  botulism,  61 

diphtheria,  75 

pyocyaneus,  92 

tetanus,  63 
Arachnoidea,  221 
Argas,  221,  226 
Ascaris,  canis,  208,  218 

lumbricoides,  208,  218 
Ascitic  fluid  (cytodiagnosis  in),  275 
Aspergillus,  concentricus,  104 

flavus,  104 

fumigatus,  104 

pictor,  105 

repens,  104 
Auchmeromyia  luteola,  228,  236 

Bacillus,  acidi  lactici,  116 

aerogenes  capsulat.,  53,  64 

anthracis,  53,  54 

anthracis  symptomat.,  53 

botulinus,  53,  60 

cloacae,  71 

coli,  78,  91,  113 

diphtheriae,  66,  73,  251,  253,  256 

dysenteriae,  78,  89 

enteritidis  (Gartner),  78,  89 

enteritidis  sporogenes,  53 

fecalis  alkaligines,  85 

icteroides,  85 

influenzae,  78,  79 

lactis  aerogenes,  91 

lepiae,  66,  70,  252,  253 

mallei,  66.  72 

mycoides,  53 

of  avian  tuberculosis,  66 

of  bovine  tuberculosis,  66 

of  chancroid,  78,  81 

of  Hofman,  76 

of  Koch- Weeks,  78,  80,  251 

of  malignant  cedema,  53,  59 

of  Morax,  78,  81 


287 


288 


INDEX. 


Bacillus  of  smegma,  66,  70 

of  timothy  grass,  66,  67 

of  trachoma  (Muller),  78 

paratyphosus  (A.  and  B.),  88 

pestis,  78,  8 1 

pneumoniae  (Friedlander),  79,  81 

prodigiosus,  93 

proteus,  89 

psittacosis,  85 

pyocyaneus,  92 

subtilis,  53 

tetani,  53,  54,  62 

tuberculosis,  66,  68 

typhosus,  79,  86,  114 

violaceus,  92 

vulgatus,  53 

xerosis,  77,  252 
Bacterium,  38 

Bacteriolytic  experiments,  128 
Balantidium  coli,  183 
Beriberi,  286 
Bile  media,  25,  268 
Bilharziasis,  199 
Blackwater  fever,  224,  286 
Blood,  color  index  of,  154 

counting  red  cells,  140 

counting  white  cells,  143 

counting  with  microscopic  field,  144 

cultures  of,  268 

differential  count  (normal),  160 

dried  films,  146 

fixation  of,  147 

fresh  preparations,  145 

making  preparations,  138 

normal  count,  154 

red  cells  of,  154 

staining  of,  148 

white  cells  of,  156 
Blood  platelets,  161 
Blood  serum,  coagulating  apparatus,  10 

preparation  of,  23 

Bordet  and  Gengou  phenomenon,  130 
Bottle  bacillus,  273 
Bouillon,  glycerine,  20 

Liebig's  extract  in,  19 

standardizing  reaction  of,  16,  17 

sterilization  of,  19 

sugar,  20,  40 

sugar  free,  20 
Broth  media,  15 
Buccal  secretions,  255 

Calliphora  vomitoria,  228,  236 
Capsule  staining,  31 


Carbol-fuchsin  stain,  28 

Cellia,  248 

Cells,  in  blood,  154,  156 

in  cytodiagnosis,  275 
Cerebrospinal  fluid,  51 

puncture  for,  276 
Cestoda,  194,  200 
Chlorosis,  154,  164 
Cholera,  94 

in  water,  114 
Chironomidse,  228,  239 
Chromatin  stains,  151,  152 
Chromogens,  92 

Chrysomyia  macellaria,  228,  237 
Chrysops,  228,  234 
Chyluria,  262 

Cladorchis  watsoni,  194,  197 
Classification,  animal  kingdom,  169 

arachnoidea,  221 

bacilli,  branching,  66 

bacilli,  gram  negative,  78 

bacilli,  spore  bearing,  53 

bacteria,  36 

cocci,  41 

flat  worms,  194 

fungi,  99 

insects,  228 

mosquitoes,  247,  248,  249 

protozoa,  173 

round  worms,  208 

spirilla,  94 

Clonorchis  endemicus,  194,  196 
Clonorchis  sinensis,  194 
Coccidiaria,  183 
Coccidium  (See  Eimeria  and  Isospora), 

185 

Coley's  fluid,  93 
Colon  bacillus,  78,  91 

in  water,  113 
Colonies,  isolation  of,  34 
Color  index,  154 
Commensalism,  171 
Complement,  122 

absorption  of,  130 

deviation  of,  129 
Conjunctival  infections,  251 
Conradi-Drigalski  medium,  26 
Cover  glasses,  2 
Cover-glass  preparations,  27 
Cryptococcus  gilchristi,  102 
Culicinae,  246 
Culture  media,  agar,  21 

bile  media,  25 

blood  agar,  24 


INDEX. 


289 


Culture,  blood  serum,  24 

bouillon,  15 

faeces  media,  25 

gelatin,  22 

litmus  milk,  23 

peptone  solution,  21 

potato,  23 

sterilization  of,  14 

sugar  bouillon,  20 
Cycloleppteron,  248 
Cystitis,  261 
Cytorrhyctes,  luis,  193 

scarlatinae,  193 

vaccinae,  193 
Cytodiagnosis,  275 

Davainea  madagascariensis,  204 
Demodex  folliculorum,  221,  224 
Dengue,  249,  286 
Dermacentor  andersoni,  227 
Dermatobia  cyaniventris,  228,  238 
Desk-microscopic,  10 
Dhobies  itch,  106 
Dibothriocephalus  latus,  204 
Dicrocoelium  lanceatum,  194,  196 
Differential  leukocyte  count,  160 
Diphtheria,  66,  73,  256 

diagnosis  of,  75 

diphtheria-like  bacilli,  76 

media  for  growing,  74 

Neisser's  stain,  31 

toxin  of,  75 
Diplococcus,  crassus,  47 

intracellular  meningitidis,  49 

lanceolat,  46 

Diplognoporus  grandis,  204 
Diptera,  228,  231 
Dipylidium  caninum,  194,  204 
Distomiasis,  195 
Double  boiler,  n 
Dum  dum  fever,  181 
Dunham's  solution,  21 
Dysentery,  amoebae  in,  175,  265 

bacilli,  89 

bacilli  in  faeces,  265 

Eberth  group,  85 
Exhinococcus  cysts,  205 
P^chinorhynchus  gigas,  219 
Ehrlich,  blood  film  method,  146 

granule  staining,  159 

tri-acid  stain,  148 
Eimeria  stiedae,  173,  185 
Endo  medium.  25 

19 


Endomyces  albicans,  101 
Endothelial  cell  in  cytodiagnosis,  275 
Entamoeba,  buccalis,  173,  176 

coli,  173,  175 

histolytica,  173,  175,  266 
Eosinophiles,  159 
Eosinophilia,  162 
Escherich  group,  85 
Eustrongylus  gigas,  208,  216 
Eye-piece  (see  ocular),  2 
Eye-strain,  3 
Eye  infections,  251 

Faeces,  263 

amoebae  in,  266 

culturing,  265 

diet  for  examination  of,  263 

fats  in,  265 

pancreatic  test,  266 

plating  media,  25 

soaps  in,  265 

Fasciola  hepatica,  194,  195 
Fasciolopsis  buski,  194,  197 
Fauces,  255 
Favus,  104 

Fermentation  tubes,  5,  10 
Films  (blood),  146 
Filter  pump,  12 
Filaria,  bancrofti,  208,  2 1 1 

demarquayi,  208,  212 

embryos,  key  to,  213 

loa,  208,  211,  252 

medinensis,  209 

ozzardi,  213 

perstans,  208,  212 

philippinensis,  213 

powelli,  213 
Fixation,  blood  films,  147 

tissues,  279 
Flagella  staining,  32 
Flagellata,  176 
Flat  worms,  194 
Fleas,  232 

key  to,  232 
Flukes,  194 

of  blood,  198 

of  intestines,  197 

of  liver,  195 

of  lungs,  198 

Focus,  microscopical,  3,  4 
Foot  and  mouth  disease,   285 
Friedlander  group,  79,  81,  252 
Fungi,  Achorion,  104 

Ascomycetes,  100 


2  go 


INDEX. 


Fungi,  Aspergillus,  104 
classification  of,  99 
Crytococcus,  102 
cultivation  of,  107 
diagnosis  of.  107 
Discomyces  bovis,  106 
Discomyces  madurae,  106 
Hyphomycetes,  106 
Madurella  mycetomi,  106 
Malassezia  furfur,  106 
Microsporoides,  106 
Microsporum  audouini,  106 
Mucor,  100 
Penicillium,  104 
Saccharomycetes,  101 
Trichophyton,  103 
Trichosporum  giganteum,  107 

Gartner  group,  85 
Gastric  contents,  270 
Gastrodiscus  hominis,  194,  197 
Gelatin,  22 

liquefaction  of,  39 

General   paralysis  (spinal  fluid  in),  276 
Giemsa's  stain,  151 
Glanders,  66,  72 
Glassware,  cleaning  of,  8 
Glossina  palpalis,  228,  236 
Gonococcus,  41,  48,  251 
Gonorrhoea,  48 
Goundou,  286 
Grabhamia,  250 
Gram  method,  28,  38 

negative  bacteria,  29 

positive  bacteria,  29 

solution,  29 

Granular  degeneration  (red  cells,)   155 
Granules  (white  cells,)  157 

Haemacytometer,  136,  137,  140 
Haemadipsa  ceylonica,  220 
Haematopota,  228,  234 
Haematoxylin  stain,  152,  286 
Haemoglobin  estimation,  138 
Haemoglobinometers,  Miescher's  138 

Sahli's,  139 

Tallquist,  139 

Haemolytic  experiments,  128 
Haemosporidia,  153,  185 
Haffkine,  cholera  vaccine,  97 

plague  prophylactic,  84 
Halzoun,  196,  237 
Hemokonia,  161 
Herpetomonas,  182 


Heterophyes  heterophyes,  194,  197 
Hirudo,  medicinalis,  208,  219 

nilotica,  208,  219 
Hodgkin's  disease,  168 
Hook  worms,  216 
Hydatid  disease,    205 
Hydrocele  agar,  24 
Hymenolepis,  nana,  194,  203 

diminuta,  203 
Hyphomycetes,  106 
Hypoderma  diana,  228,  238 

Immersion  objectives,  3 
Immune  sera,  antimicrobic,  120 

antitoxic,  120 

diphtheria,  15 

in  diagnosis,  124 

preparation,  124,  125 

tetanus,  63 
Immunity,  active,  120 

artificial,  119 

natural,  119 

passive,  120 
Incubators,  body  temperature,  n 

electrical,  n,  12 

petroleum  lamp,  n 

room  temperature,  12 
Indol,  test  for,  21 
Influenza,  79 
Infusoria,  183 
Inoculation  animals  (tuberculosis),  67 

animals  (plague),  84 

of  media,  36 
lodophilia,  152 
Insecta,  228 

Isospora  bigemina,  173,  185 
Itch  mite,  223 
Ixodidae,  221,  224,  226 

Japanese  river  fever,  202,  286 
Joints,  gonococcus  in,  49 

Kala  azar,  181 

Kedani  mite,  222,  286 

Key  to  branching,  curving  bacilli,  66 

to  cocci,  41 

to  filarial  embryos,  213 

to  fleas,  232 

to  Gram  negative  bacilli,  78 

to  spirilla,  94 

to  spore-bearing  bacilli,  53 

Lactic-acid  bacteria,  116 
Lamblia  intestinalis,  173,  182 


INDEX. 


2QI 


Leeches,  219 
Leishmania,  181 

donovani,  173,  181 

tropica,  173,  181 
Leprosy,  70 

diagnosis  of,  71 

in  rats,  71 
Leukaemia,  166 

lymphatic,  168 

splenomyelogenous,  167 
Leukocytosis,  162 
Leukopenia,  161 
Leydenia  gemmipara,  173,  176 
Light  in  microscopical  work, 
Linguatula  rhinaria,  221,  227,  254 
Liquefaction  of  gelatine,  39 
Litmus,  23 
Liver  abscess,  163 
Loeffler  serum,  24,  256 

stain,  28 

Lumbar  puncture,  276 
Lymphocytosis,  163 
Lymphocytes,  large,  157 

small,  157 

Madura  foot,  106 
Magnifying  power,  137 

of  oculars,  2 
Malaria,  185 

diagnosis  of,  192 

differential  tables,  191,  192 

life  cycle,  189 

life  history,  187 

Romanowsky  stain  in,  152 
Mallein,  72 

Malta  fever,  51,  261,  269 
Mansonia,  249 
Mast  cells,  159 
Measles,  285 
Megarhininae,  248 
Meat  poisoning,  60,  61 

toxin  of,  6 1 
Malignant  pustule,  55 
Mechanical  stage,  i 
Media  (see  culture  media),  25 
Megaloblast,  156 
Melaniferous  leukocytes,  164 
Meningococcus,  49,  276 
Micrococcus,  41 

catarrhalis,  41,  51,  253 

melitensis,  41,  51 

tetragenus,  41,  44,  253 
Mi(  rometer  disk,  2,  135 

standardization  of,  136 


Micrometer  screw,  3 

Micrometry,  2,  135 

Microscope,  i 

Microscopical  sections  (see  tissue),  281 

quick  diagnostic  method,  281 
Milk,    bacteriological    examination    of, 

"5 

lactic-acid  bacteria  in,  116 

leukocytes  in,  115 
Mites,  221,  222,  223 
Mosquitoes,  anatomy  of,  240 

classification  of,  247,  248,  249 

dissection  of,  244 

larvae  of,  242 

ova  of,  242    i 

pupae  of,  244 
Moulds,  (see  fungi),  99 
Mononuclear  leukocytes,  158 
Motility,  36 

Brownian,  37 

current,  37 
Mucidus,  249 
Mumps,  285 

Musca  domestica,  228,  235 
Mutualism,  170 
Myelocytes,  160 
Myzomyia,  248J 
Myzorhynchus,  248 

Nasal  infections,  diphtheria  in,  253 

leprosy  in,  253 

Necator  americanus,  208,  218 
Negri  bodies,  277 
Neisser's  stain,  31,  256 
Nematoda,  208 

Nomenclature,  in  animal  parasitology, 
171 

law  of  priority  in,  171 
Normob  lasts,  156 
Normal  solutions,  284 
Notes,  blank,  bacteriology,  134,  135 

blood  work,  168,  169 

parasitology,  animal,  250,  251 
Numerical  aperture,  3 
Nyssorhynchus,  248 

Ocular  infections,  251 

animal  parasites  in,  252 

bacilli  in,  252 

gnococcus  in,  252 

M.  catarrhalis  in,  51 

pneumococcus  in,  251 
Objectives,  i,  2 
Oculars,  i,  2 


2Q2 


INDEX. 


Opisthorchis,  felineus,  194,  197 

sinensis,  197 
Opsonic  power,  119 

apparatus  in,  13 

determination  of,  131 
Ornithodoros,  221,  226 
Oxyuris  vermicularis,  208,  219 

Pangonia,  228,  234 

Paragonimus  westermani,  194,  198 

Parasitism,  170 

Pasteurelloses,  82 

Pediculus  capitis,  228,  229 

Pediculus  vestimenti,  228,  229 

Pellagra,  104, 

Penicillium  crustaceum,  104 

Petri  dishes,  7 

Pfeiffer's  phenomenon,  97,  128 

Pharyngeal  secretions,  255 

Phthirjus  pubis,  228,  230 

Piedra,  107 

Pinta,  105 

Pipettes,  bacteriological,  7,  12,  13 

capillary  bulb,  12 
Piroplasmata,  224 
Plague,  8 1 

diagnosis  of,  84 

flea  in,  84,  233 

pneumonia,  260 

prophylaxis,  260 
Platinum  wire,  n 
Pleural  fluids  (cytodiagnosis),  276 
Pneumococcus,  41,  46,  251 
Poikilocytes,  155,  165 
Polymorphonuclear  leukocytes,  159 
Porocephalus  constrictus,  227 
Protozoa,  173 
Psychodidae,  228,  240 
Pulex,  cheopis,  228,  233 

irritans,  228,  233 
Pulicidae,  228,  231 
Pus  cultures  from,  271 

tetanus  in,  272 
Pyretophorus,  248 

Rabies,  277,  285 

preservation  of  dog  in,  278 
Reaction  of  media,  37 

standardization  of,  16,  17,  18,  284 
Red  blood-cells,  counting  of,  140 

normal,  155 

nucleated  red  cells,  155 

polychromatophilia,  155 

punctate  basophilia,  155 


Relapsing  fever,  177 

Rheumatism  (acute),  285 

Rhizoglyphus  parasiticus,  222 

Rhizopoda,  173 

Rhynchota,  228,  229 

Rice  cooker,  10,  14,  15 

Ring- worms,  103 

Rocky  mountain  spotted  fever,  224, 

285 

Roetheln,  285 

Romanowsky  stains,  151,  152 
Round  worms,  208 

Sabouraud's    medium    for    moulds, 

1 08 
Saccharomyces,  anginosse,  101 

blanchardi,  101 

cerevisiae,  101 
Sarcina  lutea,  41,  43 
Sarcoptes  scabiei,  221,  223 
Sarcophaga  carnaria,  228,  237 
Sarcopsylla  penetrans,  228,  233 
Sarcosporidia,  192 
Scarlet  fever,  285 
Schistosomum  haematobium,  198 

japonicum,  199 

mansoni,  199 
Screw  worm,  23 7>  254 
Sections,  making  and  staining,  282 
Serum  (see  immune  serum),  120 
Sewage,  in  water,  109 
Shiga's  bacillus,  90 
Simulidae,  228,  240 
Skin  infections,  273 

itch  mite,  274 

leprosy  in,  274 

pus  cocci  in,  273 

sarcopsylla  in,  274 
Slides,  cleaning,  9 

concave,  9 
Smallpox,  193,  285 
Sparganum,  mansoni,  207 

prolifer,  207 
Spirillum  cholerae  asiaticae,  94,  114 

metschnikovi,  94 

of  Finkler,  Prior,  94 

tyrogenum,  94 
Spirochaeta,  177 

duttoni,  173,  178,  227 

recurrentis,  173,  177 

refringens,  173,  178 

Vincenti,  173,  178,  256 
Spores,  spore-bearing  bacilli,  53 

staining,  32 


INDEX. 


293 


Sporozoa,  183 
Sprue,  286 
Sputum,  258 

amoeba  in,  260 

centrifugalization  for  T.  B.,  259 

culturing,  259 

fixing  smears,  258 

Paragonimus  eggs  in,  260 

plague  pneumonia,  260 
Staining  methods,  27 
Stains,  acid  fast,  30 

Balch's,  149 

capsule,  31 

carbol  fuchsin,  28 

flagella,  32 

Giemsa's,  151,  283 

Gram's  method,  28 

haematoxylin,  152,  286 

King's,  150 

Leishman's,  150 

Loffler's  methylene  blue,  28 

Neisser's,  31 

Nicolle's,  282 

Smith's  formal  fuchsin,  30 

spore,  32 

tri-acid,  148 

Van  Giesen's,  282 

Wright's,  149 
Staphylococcus,  44 

epidermidis  albus,  41,  45 

pyogenes  albus,  41,  45 

pyogenes  aureus,  41,  45 
Stegomyia,  249 
Sterilization,  Arnold,  5,  15 

autoclave,  5,  6,  14 

glass  ware,  4 

hot  air,  5 

pathogenic  bacteria,  8 
Stomach  contents,  270 

Boas-Oppler  bacillus,  270 

cancer  cells  in,  270 
Stomoxys,  228,  236 
Streptococcus,  41,  42 

anginosus,  41 

capsulatus,  47 

coli  gracilis,  41 

fecal  is,  41 

pyogenes,  41,  43 

salivarius,  41 
Strong,  cholera  prophylactic,  97 

plague  vaccine,  85 
Strongylidae,  216 

Strongyloides  stercoralis,  208,  209 
Swabs,'  7 


Syphilis,  fixation  of  complement  in  di- 
agnosis, 130,  178 
Giemsa's  stain  in,  256 

Tabanus,  228,  234 
Tables,  insects,  228 

mosquitoes,  246,  248,  249 

of  arachnoids,  221 

of  flat  worms,  194 

of  fungi,  99 

of  protozoa,  173 

of  round  worms,  194 

pressure  and  temperature,  7 
Taenia,  africana,  203 

echinococcus,  205 

saginata,  194,  202 

solium,  194,  202,  252 
Taeniorhynchus,  249 
Tape  worms,  adult,  200 

somatic  or  larval,  205 
Terminology,  172 
Test-tubes,  8 
Tetanus,  53,  62 

antitoxin,  63 

diagnosis  of,  64 

toxin,  63 
Theobaldia,  249 
Thionin,  148 
Thorn-headed  worm,  219 
Throat  examination,  236 
Thrush,  101,  256 
Ticks,  224 

classification  of,  226 

life  history,  225 
Tinea,  103 

cruris,  107 

imbricata,  104 

versicolor,  106 
Tissue,  acetone  method,  280 

chloroform  method,  280 

dehydration  of,  280 

fixation  of,  279 

imbedding,  280 

preparation  of  for  sections,  279 
Titration  of  media,  17,  284 
Toisson's  solution,  141 
Toxin,  121 

diphtheria,  75 

tetanus,  63 

Transitional  leukocyte,  158 
Trematoda,  194 
Treponema,  151,  178 

pallidum,  173,  178 

pertenue,  173,  179 


294 


INDEX. 


Trichomonas  intestinalis,  173 

vaginalis,  173,  182 
Trichinella  spiralis,  208,  214 
Trichinosis,  214 

Trichophyton  mentagrophytes,  103 
Sabouraudi,  103 
tonsurans,  103 

Trichocephalus  trichiurus,  208,  213 
Trichostrongylus  instabilis,  216 
Trombidiidae,  222 

Trombidium  holosericeum,  221,  222 
Trypanoplasma,  181 
Trypanosoma,  brucei,  181 

equinum,  181 

equiperdum,  181 

evansi,  181 

gambiense,  173  and  180,  236 

lewisi,  180,  181 

Trypanosomiasis,    180,   164,   276 
Tsetse  flies,  236 
Tuberculin,  69 
Tuberculosis,  bacillus  of,  68 

diagnosis  of,  70 

in  guinea  pigs,  57 
Tubes,  fermentation,  10 

test-tubes  for  agglutination,  127 

Wright's  U-tube,  13 
Turck,  irritation  cells,  161 

ruling  on  haemacytometer,  140 
Typhoid,  agglutination  in,  87 

blood  cultures,  269 

carriers,  88 

gall  stones  in,  87 

in  water,  114 

serum  for,  87 

vaccination  in,  87 
Typhus  fever,  285 
Tyroglyphus  longior,  221,  222 


Urine,  261 

chylous,  262 
moulds  in,  261 
'schistosomum  eggs  in,  262 
smegma  bacillus  in,  261 

Vaccines,  132 

preparation  of,  132 

standardizing  of,  133 
Varicella,  285 
Vincent's  angina,  178,  256 

Water,     bacteriological    examination, 
109 

cholera  spirillum  in,  114 

colon  bacillus  in,  113 

qualitative  bacteriological  examina 
tion,  112 

quantitative  bacteriological  examin- 
ation, no 

typhoid  bacillus  in,  114 
Whip  worms,  213 
White  blood-cells,  156 

counting  of,  143 

normal  count  in  disease,  164 
Whooping  cough,  80,  285 
Widal  tests,  126 
Woolsorter's  disease,  55 
Working  distance,  3 
Wright's  blood  stain,  149,  157 

method  for  anaerobes,  59 

method  for  standardizing  vaccines, 
133 

Yaws,  179 
Yersin's  serum,  85 
Yellow  fever,  249,  286 


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Equivalent  Fahrenheit  and 
Centigrade  tables  for  the  tem- 
peratures in  common  use  in 
laboratories : 

1.  Those   employed    in   pre- 
serving biological  products  and 
post  mortem  material;   also  in 
centrifuging  experiments  to 
separate  complement   and  am- 
bcceptor      (freezing      tempera- 
tures) . 

2.  Those  for  culturing  gela- 
tin (melting-point,  25°  C.)  as  in 
water    work     (room     tempera- 
tures) . 

3.  Those    fcr    growing    im- 
portant   pathogenic    organisms 
(body  temperatures). 

4.  Those   for    pasteurization 
and     sterilization    of    bacterial 
vaccines.      Also     for     paraffin 
bath      (pasteurizing     tempera- 
tures) . 

5.  Those  for  sterilization  of 
dressings  and  media.     Also  for 
certain    disinfection    of   spore- 
bearing     bacterial     contamina- 
tion (autoclave  temperatures). 


UNITS  IN  COMMON  USE  IN  LABORATORIES. 

Cubic  Meter. — Unit  of  space  for  the  number  of  organisms  in  air. 
It  contains  1000  litres.  It  is  equal  to  1-308  cubic  yards  or 
35.316  cubic  feet.  One  thousand  cubic  feet,  the  unit  of  space 
in  disinfection,  is  equal  to  28.3  +  cubic  meters. 

Litre. — Unit  of  space  for  normal  volumetric  solutions.  It  contains 
1000  cubic  centimeters.  It  is  equal  to  1.0567  quarts  or  33.8  + 
ounces.  A  liter  of  distilled  water  weighs  i  kilogram. 

Cubic  Centimeter.— Unit  of  space  for  organisms  in  water,  milk, 
vaccines,  etc.,  ic.c.=o.27  fl.  dr.  There  are,  approximately, 
16  drops  in  i  c.c. 

Cubic  Millimeter. — Unit  of  space  for  blood-cells.  There  are  1000 
cubic  millimeters  in  i  cubic  centimeter  and  i  million  cubic  milli- 
meters in  i  litre.  In  water  analysis,  as  there  are  i  million  milli- 
grams in  one  litre,  parts  in  the  million  and  milligrammes  per 
litre  are  the  same. 

i  Meter  =  3  9.3  7  inches. 

i  Centimeter  =  .3937  inch.     Approximately,  2/5  inch. 

i  Millimeter  =  .0393  inch.     Approximately,  1/25  inches. 

i  Kilogram  =  2.2+  pounds  av. 

i  Gram  =  15. 432  grains. 

i  Milligram  =  0.0154  grain.     Approximately,  1/64  grain. 

A  pound  avoirdupois  is  equal  to  453.59  gm. 

One  hundred  cubic  centimeters  of  a  saturated  solution  contains: 


Water 

Alcohol 

Methylene  blue, 

6.68 

0.66  grams. 

Gentian  violet, 

i-75 

4.42  grams. 

Basic    fuchsia, 

0.66 

2.92  grams. 

YC  88491 


GRAM'S  METHOD  OF  STAINING. 

1.  Prepare  even  thin  film  and  fix. 

2.  Aniline  gentian  violet, -2  to  5  minutes. 

3.  Gram's  iodine  solution,  i  to  2  minutes.   (Tea-leaf  color.) 

4.  Wash  in  water  and  decolorize  with  95%  alcohol. 

5.  Wash    in    water  and    counterstain  with  Bismarck    brown  or 
with  dilute  carbol  fuchsin. 

6.  Wash,  dry  and  mount. 

Practically  all  pathogenic  cocci  are  Gram  positive,  except  the 
Gonococcus,  the  Meningococcus,  the  M.  catarrhalis  and  the  M. 
melitensis. 

Practically  all  pathogenic  bacilli  are  Gram  negative,  except  the 
e  pore-bearing  ones  (exception  B.  malig.  cedemat.),  the  acid-fast  ones 
and  diphtheria  and  diphtheroid  organisms. 


